Compositions and methods directed to treating liver fibrosis

ABSTRACT

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the COL1A1, TGF-β, and SMAD2/3 genes, and methods of using such dsRNA compositions to inhibit expression of COL1A1, TGF-β, and SMAD2/3.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pending U.S. application Ser. No. 15/905,229 filed Feb. 26, 2018, which is a continuation application of U.S. Ser. No. 13/701,551, filed Feb. 26, 2013, now U.S. Pat. No. 9,944,671, which is a 35 U.S.C. § 371 National Phase Entry Application of International Application No. PCT/US2011/038889 filed 2 Jun. 2011, which designates the U.S., and which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 61/408,271 filed on Oct. 29, 2010, U.S. Provisional Application Ser. No. 61/371,481 filed on Aug. 6, 2010, and U.S. Provisional Application Ser. No. 61/350,829 filed on Jun. 2, 2010, the contents of each of which are herein incorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 2, 2011, is named 51058PCT.txt and is 864,110 bytes in size.

FIELD OF THE INVENTION

The invention relates to the specific inhibition of the expression of the collagen1A1 (COL1A1), transforming growth factor beta 1 (TGFβ) and SMAD homolog 2 and 3 (SMAD2/3) genes in the liver, and the inhibition of hepatic fibrosis. In particular, the inhibition is directed at the genes expression in hepatic stellate cells.

BACKGROUND OF THE INVENTION

In the United States, chronic liver disease is the 12th leading cause of disease-related death, with approximately 28,000 people dying each year from chronic liver disease. In the US and Europe, liver cirrhosis is the most common cause of non-neoplastic death among hepatobilliary and digestive diseases. Furthermore, the fourth leading cause of death in urban American males is alcoholic liver disease. Chronic liver disease is characterized by a process of progressive destruction and regeneration of liver cells that leads to hepatic fibrosis and cirrhosis.

Hepatic fibrosis is a process that occurs when the liver is damaged. Such damage can be the result of viral activity (e.g., chronic hepatitis types B or C) or other liver infections (e.g., parasites, bacteria); exposure to chemicals (e.g., pharmaceuticals, recreational drugs, excessive alcohol, exposure to pollutants); autoimmune processes (e.g., autoimmune hepatitis); metabolic disorders (e.g., lipid, glycogen, cholesterol or metal storage disorders); or cancer growth (primary or secondary liver cancer). The principal cell type responsible for liver fibrosis is the hepatic stellate cell (HSC), a resident perisinusoidal cell that takes up vitamin A from circulation and stores it.

In the liver, fibrosis is not only the result of necrosis, collapse and scar formation but also the result of derangements in the synthesis and degradation of matrix by injured mesenchymal cells that synthesize the various components of the extracellular matrix (ECM). Hepatic fibrosis is a gradual process of increased secretion and decreased degradation of extracellular materials. It is believed that the process is initiated by damage to hepatic cells. Necrotic hepatic cells present after injury evoke a reaction of inflammatory cells which secrete mediators including cytokines that stimulate matrix producing cells. More particularly, the damage to hepatic cells leads to the activation and secretion of multiple cellular factors from Kupffer cells, the macrophages which line the liver sinusoids. These factors, along with the cellular factors secreted by damaged hepatic cells, thrombocytes, and endothelial cells of the hepatic sinusoid become activators of HSCs. Upon activation, the HSCs differentiate to proliferative, fibrogenic and contractile myofibroblasts, and, via self-secretion and parasecretion, the HSCs proliferate and synthesize a massive amount of ECM materials, which gradually accumulate and form fibrous masses in the liver. The myofibroblasts secrete procollagen, which accumulates as insoluble collagen after its terminal domains are cleaved by procollagen peptides, causing fibrosis. The collagen-specific chaperone, heat shock protein 47 (HSP47), facilitates collagen secretion by ensuring proper triple-helix formation of procollagen in the endoplasmic reticulum and has also been implicated in translational regulation of procollagen synthesis.

The ECM materials are of the following categories: collagens (type I, III, IV, V, VI, VII), glycoprotein (laminin, fibronectin, entactin, undulin, and elastin) and proteoglycans (chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate and heparin). These ECM materials interact with cell surface receptors and macromolecules such as growth factors, collagens, fibronectin, laminin etc. Fibrosis develops after repeated and persistent injury that overcomes the ability of the liver to degrade the ECM matrix via degrading enzymes produced by fibroblasts, neutrophils and macrophages. The excessive ECM materials built up in the liver lead to derangement of the hepatic architecture and portal hypertension, and can produce the irreversible and detrimental rearrangement of the liver circulation known as cirrhosis. Cirrhosis is characterized by replacement of liver tissue by fibrous, scar tissue and regenerative nodules. These regenerative nodules are the result of a process in which damaged tissue tries to regenerate and this leads to the loss of liver function. Therefore, fibrosis is both a sign of liver damage and a major contributor to liver failure via progressive cirrhosis of the liver. Since fibrosis is a common development in a variety of chronic liver diseases, treatment and/or prevention of liver fibrosis is of great importance.

SUMMARY OF THE INVENTION

Described herein are compositions and methods that effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a collagen1A1 (COL1A1), transforming growth factor beta 1 (TGFβ) and/or SMAD homolog 2 and 3 (SMAD2/3) genes, in a cell or mammal. Also described are compositions and methods for treating and/or preventing liver fibrosis caused by the overexpression of a COL1A1 gene that occurs as a result of damage to liver cells, including, but not limited to, chronic liver disease. Liver fibrosis is a result of a viral or other infection, an autoimmune disorder, a bile duct obstruction, metabolic disorders, alcohol abuse, primary biliary cirrhosis, non-alcoholic steatohepatitis (NASH), exposure to chemicals, and cancer among others.

The embodiments of compositions and methods for treating and/or preventing chronic liver disease and hepatic fibrosis are based, at least in part, on the premise that if at least one of the extracellular matrix (ECM) materials that are produced in excess and are contributory to fibrosis during liver damage, including damage occurring under chronic liver injury conditions, can be reduced, then fibrosis in the liver can be, at a minimum, reduced if not prevented. The compositions and methods are based on RNA interference of gene expression of relevant ECM genes and also of the signaling molecules, e.g., TGF-β and SMAD2/3, which are involved in the activation of hepatic stellate cells (HSCs), which are the major producers of ECM materials during the fibrosis process.

As used herein, the term “iRNA” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein inhibits COL1A1, TGFβ or SMAD2/3 gene expression in a cell or mammal.

The iRNAs included in the compositions featured herein encompass a dsRNA having an RNA strand (the antisense strand) having a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of a COL1A1, TGFβ or SMAD2/3 gene.

In one embodiment, an iRNA for inhibiting expression of a COL1A1, TGFβ or SMAD2/3 gene includes at least two sequences that are complementary to each other. The iRNA includes a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand includes a nucleotide sequence that is substantially complementary to at least part of an mRNA encoding COL1A1, TGFβ or SMAD2/3 and the region of complementarity is 30 nucleotides or less, and at least 15 nucleotides in length. Generally, the iRNA is 19 to 24, e.g., 19 to 21 nucleotides in length. In some embodiments the iRNA is from about 15 to about 25 nucleotides in length, and in other embodiments the iRNA is from about 25 to about 30 nucleotides in length. The iRNA, upon contacting with a cell expressing COL1A1, TGFβ and/or SMAD2/3, inhibits the expression of a COL1A1, TGFβ or SMAD2/3 gene respectively by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more, such as when assayed by a method as described herein. In one embodiment, the COL1A1iRNA, TGFβiRNA or SMAD2/3iRNA is formulated in a stable nucleic acid lipid particle (SNALP).

In one embodiment, an iRNA featured herein includes a first sequence of a dsRNA that is selected from the group consisting of the sense sequences of Tables 3-7 and a second sequence that is selected from the group consisting of the corresponding antisense sequences of Tables 3-7. The iRNA molecules featured herein can include naturally occurring nucleotides or can include at least one modified nucleotide, including, but not limited to a 2′-O-methyl modified nucleotide, a nucleotide having a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative. Alternatively, the modified nucleotide can be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Generally, such a modified sequence will be based on a first sequence of iRNA selected from the group consisting of the sense sequences of Tables 3-7, and a second sequence selected from the group consisting of the corresponding antisense sequences of Tables 3-7.

In one embodiment, an iRNA as described herein targets a COL1A1 RNA transcript, and in another embodiment, the iRNA targets a TGFβ RNA transcript. In yet another embodiment, the iRNA targets a SMAD2 transcript. In yet another embodiment, the iRNA targets a SMAD3 transcript.

In some embodiments of the aspects described herein, the iRNA is a dsRNA which comprises the RNA sequence pair (AD-21349-b1) of SEQ ID NO: 53 (41100-b1D) and SEQ ID NO: 54 (41101-b1D), the RNA sequence pair (AD-22138) of SEQ ID NO: 69 (42861) and SEQ ID NO: 70 (42862), the RNA sequence pair (AD-22149) of SEQ ID NO: 91 (42883) and SEQ ID NO: 92 (42884) or the RNA sequence pair (AD-20916-b1) of SEQ ID NO: 140 (40322-b1) and SEQ ID NO: 141 (40323-b1).

In one embodiment, an iRNA featured in the invention targets a non-coding region of a COL1A1, TGFβ or SMAD2/3 RNA transcript, such as the 5′ or 3′ untranslated region.

In one aspect, embodiments of the invention provide a cell containing at least one of the iRNAs featured in the invention. The cell is generally a mammalian cell, such as a human cell. In one preferred embodiment, the cell is a hepatic cell. In a more preferred embodiment, the cell is a HSC.

In another aspect, embodiments of the invention provide a pharmaceutical composition for inhibiting the expression of a COL1A1, TGFβ and/or SMAD2/3 gene in an organism, preferably a human subject. The composition typically includes one or more of the iRNAs described herein and a pharmaceutically acceptable carrier or delivery vehicle. In one embodiment, the composition is used for treating liver disease, including, but not limited to, a chronic liver disease. In one embodiment, the composition is used for treating hepatic fibrosis. In some embodiments, the liver disease or hepatic fibrosis is a result of a viral or other infection, an autoimmune disorder, a bile duct obstruction, metabolic disorders, alcohol abuse, primary biliary cirrhosis, NASH, exposure to chemicals, or cancer.

In another embodiment, the pharmaceutical composition is formulated for administration in a dosage regimen described herein, e.g., not more than once every four weeks, not more than once every three weeks, not more than once every two weeks, or not more than once every week. In another embodiment, the administration of the pharmaceutical composition can be maintained for a month or longer, e.g., one, two, three, or six months, or one year, or five years, or ten years, or longer, including the remaining lifetime of a subject, e.g., for a subject having a chronic liver disease or condition.

In another embodiment, a composition containing an iRNA featured described herein, e.g., a dsRNA targeting COL1A1, is administered with a non-iRNA therapeutic agent, such as an agent known to treat the underlying cause of the liver disease or hepatic fibrosis, e. g. a viral or other infection, an autoimmune disorder, a bile duct obstruction, metabolic disorders, alcohol abuse, primary biliary cirrhosis, NASH, exposure to chemicals, and cancer. In another embodiment, a composition containing an iRNA featured described herein, e.g., a dsRNA targeting COL1A1, is administered along with a non-iRNA therapeutic regimen, such as an anti-hepatitis C virus agent. For example, an iRNA featured in the invention can be administered along with an interferon alpha for treatment of hepatitis C infection.

In another embodiment, a COL1A1 iRNA, TGFβ iRNA and/or SMAD2/3 iRNA is administered to a patient, and then the non-iRNA agent or therapeutic regimen is administered to the patient (or vice versa). In another embodiment, a COL1A1 iRNA, TGFβ iRNA and/or SMAD2/3 iRNA and the non-iRNA therapeutic agent or therapeutic regimen are administered at the same time. In one embodiment, the therapeutic agent is, for example, an interferon alpha for the treatment of hepatitis C infection. In another embodiment, a combination of more than one iRNA is adminstered to the patient. For example, a combination of COL1A1 iRNA and TGFβ iRNA, a combination of COL1A1 iRNA and SMAD2/3 iRNA or a combination of COL1A1 iRNA, TGFβ iRNA and SMAD2/3 iRNA.

In another aspect, provided herein is a method for inhibiting the expression of a COL1A1, a TGFβ, and/or a SMAD2/3 gene in a cell by performing the following steps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid         (dsRNA), wherein the dsRNA includes at least two sequences that         are complementary to each other. The dsRNA has a sense strand         having a first sequence and an antisense strand having a second         sequence; the antisense strand has a region of complementarity         that is substantially complementary to at least a part of an         mRNA encoding COL1A1, TGFβ, and/or a SMAD2/3 and where the         region of complementarity is 30 nucleotides or less, i.e., 15-30         nucleotides in length, and generally 19-24 nucleotides in         length, and where the dsRNA, upon contact with a cell expressing         COL1A1, TGFβ, and/or a SMAD2/3 inhibits expression of a COL1A1,         TGFβ, or SMAD2/3 gene respectively by at least 10%, preferably         at least 20%, at least 30%, at least 40% or more; and     -   (b) maintaining the cell produced in step (a) for a time         sufficient to obtain degradation of the mRNA transcript of the         COL1A1, TGFβ, or SMAD2/3 gene, thereby inhibiting expression of         a COL1A1, TGFβ, or SMAD2/3 gene respectively in the cell.

In another embodiment, the method is for inhibiting target gene expression in a liver cell, preferably in a hepatic stellate cell.

In other aspects, provided herein are methods for treating, preventing, reversing or managing liver disease or hepatic fibrosis mediated by, in part, COL1A1 overexpression. In one embodiment, the method includes administering to a patient in need of such treatment, prevention, reversal or management a therapeutically or prophylactically effective amount of one or more of the iRNAs featured herein. In some embodiments, the patient has a chronic liver disease caused by a viral or other infection, an autoimmune disorder, a bile duct obstruction, metabolic disorders, alcohol abuse, primary biliary cirrhosis, NASH, exposure to chemicals, or cancer. In another embodiment, administration of the iRNA targeting COL1A1, TGFβ, or SMAD2/3 alleviates or relieves the severity of at least one symptom of a liver disease or hepatic fibrosis, in the patient, such as high serum albumin and/or jaundice.

In one aspect, the invention provides a vector for inhibiting the expression of a COL1A1, TGFβ, and/or a SMAD2/3 gene in a cell. In one embodiment, the vector includes at least one regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of an iRNA as described herein.

In another aspect, the invention provides a cell containing a vector for inhibiting the expression of a COL1A1, TGFβ, and/or a SMAD2/3 gene in a cell. The vector includes a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the iRNAs as described herein.

In yet another aspect, the invention provides a composition containing a COL1A1iRNA, in combination with a second iRNA targeting a second gene that is involved in signaling pathway leading to the excess deposition of collagen extracellularly in the liver. For example, the second gene can be the gene encoding TGFβ, SMAD2 or SMAD3.

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the efficacy of single dose collagen1A1 siRNA in NIH-3T3 cells in vitro. siRNA AD-21349 (SEQ ID NO: 53 and SEQ ID NO: 54), has an IC₅₀ of ˜65 pM on the production of COL1A1 mRNA.

FIG. 2 demonstrates a treatment regimen of a CCl₄ mouse model in which the AF09 and AF12-based delivery of siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54) was evaluated.

FIG. 3 demonstrates efficacy of COL1A1 knock-down in a CCl₄ mouse model using AF09 and AF12-based delivery of siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

FIG. 4 demonstrates a treatment regimen of a CCl₄ mouse model in which the dosage response of the AF09 and AF12-based delivery of siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54) was evaluated.

FIG. 5 demonstrates dose dependent efficacy of COL1A1 knock-down in a CCl₄ mouse model using the AF12-based delivery of siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

FIG. 6 shows a treatment regimen of a CCl₄ mouse model in which AF11-based delivery of siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54) was evaluated.

FIG. 7 demonstrates efficacy of COL1A1 knock-down in a CCl₄ mouse model using AF11-based delivery of siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

FIG. 8 demonstrates efficacy of ACTA2 knock-down in a CCl₄ mouse model using AF12-based delivery of siACTA2.

FIG. 9 shows efficacy of a single dose TGFβ siRNA (10 nM) in NIH-3T3 cells in vitro. The TGFβ siRNAs AD22149 (SEQ ID NO: 91 and SEQ ID NO: 92) and AD22138 (SEQ ID NO: 69 and SEQ ID NO: 70) have an IC₅₀ of ˜20 pM and ˜70 pM respectively on TGFβ expression.

FIG. 10 shows efficacy of a single dose SMAD2 siRNA (10 nM) in NIH-3T3 cells in vitro. The siRNA AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141) has an IC₅₀ of ˜75 pM on SMAD2 expression.

FIG. 11 shows efficacy of SMAD2 knock-down in a CCl₄ mouse model using lipid nanoparticle-based delivery of siSMAD2 AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141).

FIG. 12 shows effects of SMAD2 knock-down on the expression of COL1A1 in a CCl₄ mouse model using lipid nanoparticle-based delivery of siSMAD2 AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141). SMAD2/3 siRNA leads to decrease in Col1a1 levels (˜50-60% from Luc siRNA injected animals) and the data demonstrates that SMADs are involved in TGFb signaling in fibrosis development

FIG. 13 shows efficacy of COL1A1 knockdown in a chronic liver injury model using a lipid nanoparticle-based formulation of siCOL1A1.

FIG. 14 shows efficacy of delivery to stellate cells with alpha-smooth muscle actin (α-SMA) in a CCl₄ mouse model using AF12-based delivery of siACTA2.

FIGS. 15A-15C demonstrate Col1a1 knockdown is mediated by RNAi. FIG. 15A shows a scheme for 5′RACE assay. FIG. 15B shows animals injected with a lipid nanoparticle-siCol1a1 formulation and sacrificed after 24 hours. FIG. 15C shows an agarose gel showing DNA bands for nested PCR.

FIG. 16 demonstrates experimental design and efficacy of COL1A1 knock-down in a bile duct ligation mouse model using AF12-based delivery of siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

FIG. 17 shows relative Col1a1 expression in bile duct ligated animals.

FIG. 18 demonstrates efficacy of COL1A1 knock-down in a bile duct ligation mouse model using siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

FIG. 19 demonstrates experimental design and efficacy of COL1A1 knock-down in a TAA mouse model of liver injury using siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

DETAILED DESCRIPTION OF THE INVENTION

Aspects and embodiments described herein relate to compositions and methods for the treatment and/or prevention of liver disease and hepatic fibrosis, including, but not limited to, hepatic fibrosis associated with chronic liver disease. The embodiments exploit the premise that if at least one of the extracellular matrix (ECM) materials that are produced in excess and are contributory to hepatic fibrosis during liver damage can be reduced, then fibrosis of the liver can be, at a minimum, reduced, if not prevented. The compositions and methods described herein are based on RNA interference with gene expression of relevant ECM genes and also those of signaling molecules, e. g. TGF-β and SMAD2/3, that are involved in the activation of hepatic stellate cells (HSCs) during the fibrotic process.

Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in Caenorhabditis elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.

One such ECM constituent that contributes to fibrosis in the liver, during chronic liver disease conditions, is collagen1A1 (COL1A1). As described herein, the inventors show that lipid nanoparticle (LNP) mediated delivery of siRNAs to HSCs in vivo significantly reduced COL1A1 expression in activated HSCs in a carbon tetrachloride (CCl4) induced mouse liver injury model (FIGS. 2-3). Following CCl₄ treatment, siRNAs formulated in LNPs comprising the cationic lipid C12-200 (PNAS, 2010, 107(2):1864) were administered via intravenous injection. Silencing of the expression of the HSC-specific target, COL1A1, demonstrated effective siRNA delivery to HSCs. The COL1A1 knock-down was dose dependent with an IC₅₀ of approximately 0.1 mg/kg (FIGS. 4-5). The inventors further surprisingly showed that effective and significant reduction of COL1A1 expression can also be achieved in a mouse model of chronic liver disease, in which mice were subjected to multiple (×6) liver insults over a 39 day period, using the COL1A1 siRNA formulations described herein (FIG. 13).

Signaling molecules involved in the activation of HSCs during the fibrotic process include, but are not limited to, TGF-β and SMAD2/3. The inventors demonstrate that LNP-mediated delivery of siRNAs targeting specifically TGF-β and SMAD2/3 significantly reduced the expression of TGF-β and SMAD2/3 in vitro (FIGS. 9-10). The inventors further demonstrate that LNP-mediated delivery of siRNAs targeting specifically SMAD2/3 in the liver significantly reduced the expression of SMAD2/3 and COL1A1 in the CCl₄ induced mouse liver injury model (FIGS. 11-12).

Fibrosis is a common pathological feature of many chronic organ failure diseases, including chronic liver disease. Necrotic cells after injury evoke reaction of inflammatory cells which secrete mediators such as cytokines which stimulate matrix producing cells. Essentially, damaged liver cells activate HSCs that secrete materials (e.g., collagens) into the matrix outside the cells. Inappropriate collagen deposition in the ECM is the hallmark of fibrosis and it occurs in the late stage of pathogenesis. Stopping fibrosis can stabilize liver function, delaying time to transplant or providing a survival benefit.

The biology of fibrosis is very well understood. HSC play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from “quiescent” HSC to fibrogenic myofibroblasts. It is a TGF-β driven process, and SMAD2 and SMAD3 function as TGFβ signaling intermediates. TGF-β, acting both directly and indirectly, is a critical mediator of this process. One approach of treating and/or preventing liver fibrosis is to inhibit HSC proliferation and/or promote HSC apoptosis. Another is to inhibit the HSCs from producing and secreting the ECM components such as collagen to the hepatic ECM that forms the fibrotic mass. COL1A1 is normally present at very low levels in liver but is up-regulated and produced by activated HSCs after insult to the liver. RNAi inhibition of expression of COL1A1, TGFβ, TGFβ-signaling intermediates, such as SMAD2 and SMAD 3, and other proteins/factors that contribute to COL1A1 overexpression can be clinically relevant in treating hepatic fibrosis, regardless of etiology.

Accordingly, aspects and embodiments described herein include iRNAs and methods of using them for inhibiting the expression of a COL1A1, TGFβ and/or SMAD2/3 gene in a cell or a mammal where the iRNA targets a COL1A1, TGFβ and/or SMAD2/3 gene respectively. Also provided herein are compositions and methods for treating and/or preventing liver fibrosis by inhibiting the expression of a COL1A1, TGFβ and/or SMAD2/3 gene. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

In one embodiment of the aspects, provided herein is a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of COL1A1, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 53 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding antisense nucleotide sequence of SEQ ID NO: 54.

In another embodiment of the aspects, provided herein is a dsRNA for inhibiting expression of TFGβ, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 69 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding antisense nucleotide sequence of SEQ ID NO: 70.

In another embodiment of the aspects, provided herein is a dsRNA for inhibiting expression of TFGβ, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 91 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding antisense nucleotide sequence of SEQ ID NO: 92.

In another embodiment of the aspects, provided herein is a dsRNA for inhibiting expression of SMAD2/3, wherein the dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 140 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the corresponding antisense nucleotide sequence of SEQ ID NO: 141.

In some embodiments of the aspects described herein, the iRNA is a dsRNA which comprises the RNA sequence pair (AD-21349-b1) of SEQ ID NO: 53 (41100-b1D) and SEQ ID NO: 54 (41101-b1D), the RNA sequence pair (AD-22138) of SEQ ID NO: 69 (42861) and SEQ ID NO: 70 (42862), the RNA sequence pair (AD-22149) of SEQ ID NO: 91 (42883) and SEQ ID NO: 92 (42884) and/or the RNA sequence pair (AD-20916-b1) of SEQ ID NO: 140 (40322-b1) and SEQ ID NO: 141 (40323-b1).

In some embodiments, the sequences for the iRNA comprise sequences identified by the sequence identifiers found in Tables 3-7. Tables 3-7 comprise sequence identifiers (SEQ ID NOs) for sense and corresponding antisense strands. Each row of Table 3 (SEQ ID NO: 1-SEQ ID NO: 62) identifies two separate iRNAs, i.e., two separate pairs of sense strands (odd number sequence identifiers of SEQ ID NO: 1-SEQ ID NO: 62) and corresponding antisense strands (even number sequence identifiers of SEQ ID NO: 1-SEQ ID NO: 62), targeting COL1A1 for use in the compositions and methods described herein. Similarly, each row of Table 4 (SEQ ID NO: 63-SEQ ID NO: 130) identifies two separate iRNAs, i.e., two separate pairs of sense strands (odd number sequence identifiers of SEQ ID NO: 63-SEQ ID NO: 130) and corresponding antisense strands (even number sequence identifiers of SEQ ID NO: 63-SEQ ID NO: 130), targeting TGF-β for use in the compositions and methods described herein. Each row of Table 5 (SEQ ID NO: 132-SEQ ID NO: 167) identifies two separate iRNAs, i.e., two separate pairs of sense strands (even number sequence identifiers of SEQ ID NO: 132-SEQ ID NO: 167) and corresponding antisense strands (odd number sequence identifiers of SEQ ID NO: 132-SEQ ID NO: 167), targeting SMAD2/3 for use in the compositions and methods described herein. Each row of Table 6 (SEQ ID NO: 185-SEQ ID NO: 2792) identifies two separate iRNAs, i.e., two separate pairs of sense strands (odd number sequence identifiers of SEQ ID NO: 185-SEQ ID NO: 2792) and corresponding antisense strands (even number sequence identifiers of SEQ ID NO: 185-SEQ ID NO: 2792) targeting COL1A1 for use in the compositions and methods described herein. Each row of Table 7 (SEQ ID NO: 2793-SEQ ID NO: 2860) identifies two separate iRNAs, i.e., two separate pairs of sense strands (even number sequence identifiers of SEQ ID NO: 2793-SEQ ID NO: 2860) and corresponding antisense strands (odd number sequence identifiers of SEQ ID NO: 2793-SEQ ID NO: 2860) targeting SMAD2/3 for use in the compositions and methods described herein. Accordingly, in some embodiments, the iRNA is a dsRNA which comprises an RNA sequence pair (“sense and corresponding antisense duplex”) or “sense strand and corresponding antisense strand” as identified by sequence identifiers (SEQ ID NO) listed in Tables 3-7 and provided herein.

The iRNAs of the compositions featured herein comprise an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a COL1A1, TGFβ and/or SMAD2/3 gene. The use of these iRNAs enables the targeted degradation of mRNAs of genes that are implicated in the fibrotic process associated with COL1A1 expression or overexpression in the liver. Very low dosages of COL1A1, TGFβ and/or SMAD2/3 iRNAs in particular can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a COL1A1, TGFβ and/or SMAD2/3 gene respectively. Using cell-based assays and in vivo model systems, the present inventors have demonstrated that iRNAs targeting COL1A1, TGFβ and/or SMAD2/3 can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a respective gene. The inventors have also surprisingly discovered that iRNAs targeting COL1A1 can be used to mediate significant reduction of COL1A1 expression under conditions of repeated or chronic liver injury. Also the inventors demonstrate that SMAD2/3 RNAi mediated inhibition of SMAD2/3 expression indirectly led to the inhibition of COL1A1 expression. Thus, methods and compositions including these iRNAs are useful for treating and/or preventing liver disease and/or liver fibrosis by directly or indirectly down regulating COL1A1 production. The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a COL1A1, TGFβ and/or SMAD2/3 gene, as well as compositions and methods for treating and/or preventing, and possibly reversing, liver disease characterized by liver fibrosis caused by the expression or overexpression of COL1A1.

Embodiments of compositions featured herein also include an iRNA having an antisense strand having a region of complementarity which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of a COL1A1, TGFβ or SMAD2/3 gene.

Embodiments of the pharmaceutical compositions featured herein include an iRNA having an antisense strand comprising a region which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of an RNA transcript of a COL1A1, TGFβ and/or SMAD2/3 gene, together with a pharmaceutically acceptable carrier.

Accordingly, in some aspects, pharmaceutical compositions containing a COL1A1 iRNA, a TGFβ iRNA and/or a SMAD2/3 iRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of a COL1A1, a TGFβ, a SMAD2 and/or a SMAD3 gene, and methods of using the pharmaceutical compositions to treat and/or prevent possibly reverse liver fibrosis caused by the overexpression of a COL1A1 gene are featured in the invention.

In one embodiment, the pharmaceutical composition comprises more than one iRNA type, i. e. iRNAs targeting more than one gene target. For example, the pharmaceutical composition comprises a combination of COL1A1 iRNA and TGFβ iRNA, a combination of COL1A1 iRNA and SMAD2/3 iRNA or a combination of COL1A1 iRNA, TGFβ iRNA and SMAD2/3 iRNA.

In one embodiment, the iRNA is delivered using a vitamin-A-coupled liposome. The HSCs in the liver take up vitamin A from circulation and store it. The Vitamin-A-coupled liposomes function to specifically target the iRNA to the Vitamin A-sequestering HSC.

In one embodiment, the lipid nanoparticle (LNP)-formulation for delivery of iRNA described herein are coupled to vitamin A in order to target the LNP-encapsulated iRNA to the vitamin A-sequestering HSC.

In some embodiments, the pharmaceutical compositions and/or treatment therapies described herein further comprise an additional iRNA targeting a collagen-specific chaperone, heat shock protein 47 (HNP47). Sato, Y. et al., (2008, Nature Biotechnology, 26:431-442) showed that inhibiting expression of HSP47 with an siRNA-bearing vitamin A-coupled liposome almost completely resolved liver fibrosis and prolonged survival in rat treated with lethal dimethylnitrosamine.

I. Definitions

For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.

As used herein, the terms “chronic liver disease” or “chronic liver condition” refer to those diseases in which the liver is subjected to repeated insults and/or injuries over a long-term period, resulting in a process of progressive destruction and regeneration of the liver parenchyma leading to fibrosis and cirrhosis. Chronic liver disease can occur as a consequence of, or is associated with, any of a variety of etiologies, including, but not limited to, viral causes (e.g., Hepatitis B, Hepatitis C, Cytomegalovirus (CMV), Epstein Barr Virus (EBV)); repeated use of toxic substances and/or drugs (e.g., alcoholic liver disease, amiodarone, methotrexate, nitrofurantoin); metabolic/genetic causes/conditions (e.g., non-alcoholic fatty liver disease, haemochromatosis, Wilson's disease); and autoimmune disorders (e.g., autoimmune chronic hepatitis; primary biliary cirrhosis; primary sclerosing cholangitis). A liver disease or condition is considered to be ‘chronic,’ as used herein, when the condition lasts or continues for at least one month, though, in some preferred embodiments, ‘chronic’ can be defined as a period of 12 weeks or greater (e.g., such as the remaining lifetime of a subject).

As used herein, the term “fibrosis” refers to the formation of fibrous tissue as a reparative or reactive process, rather than as a normal constituent of an organ or tissue. Fibrosis is characterized by fibroblast accumulation and collagen deposition in excess of normal deposition in any particular tissue. Fibrosis occurs as the result of inflammation, irritation, or healing. As used herein the term “fibrosis” is used synonymously with “fibroblast accumulation and collagen deposition”. Fibroblasts are connective tissue cells, which are dispersed in connective tissue throughout the body. Fibroblasts secrete a nonrigid ECM containing type I and/or type III collagen. In response to an injury to a tissue, nearby fibroblasts migrate into the wound, proliferate, and produce large amounts of collagenous extracellular matrix. Collagen is a fibrous protein rich in glycine and proline that is a major component of the extracellular matrix and connective tissue, cartilage, and bone. Collagen molecules are triple-stranded helical structures called α-chains, which are wound around each other in a ropelike helix. Collagen exists in several forms or types; of these, type I, the most common, is found in skin, tendon, and bone; and type III is found in skin, blood vessels, and internal organs.

As used herein, the term “fibrotic process” refers to the temporal and progressive deposition of fibrous tissue during fibrosis.

As used herein, the terms “hepatic fibrosis” refers to the fibrosis present and/or occurring in the liver. The term “hepatic fibrosis” and “liver fibrosis” are used interchangeably.

As used herein, the term “cirrhosis” refers to a late stage of hepatic fibrosis where the liver experiences loss of functional liver cells. Normal liver tissue is replaced with fibrous tissue resulting in widespread distortion of normal hepatic architecture. A major characteristic is regenerative nodules surrounded by dense fibrotic tissue. The overgrowth of fibrosis scar tissue inhibits the liver's proper functioning Cirrhosis is usually considered irreversible.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured herein by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods described herein.

As used herein, the term “iRNA” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein inhibits the expression of COL1A1, TGFβ, or SMAD2/3 gene in a cell.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a COL1A1, a TGFβ, a SMAD2 or a SMAD3 gene, including messenger (mRNA) that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs (bp), while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” an mRNA refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding COL1A1). For example, a polynucleotide is complementary to at least a part of a COL1A1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding COL1A1.

The term “double-stranded RNA” or “dsRNA,” as used herein refers to an iRNA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 36 base pairs (bp) in length, e.g., 15-30 bp in length. Considering a duplex between 9 and 36 bp the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp, 19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 20-30 bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp, 21-23 bp, or 21-22 bp. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 bp in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.” The term “siRNA” is also used herein to refer to a dsRNA as described above.

The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleoside, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.

In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term “iRNA.”

In one aspect, an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, in one embodiment, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO2009082817. Examples of “SNALP” formulations are described elsewhere herein.

“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA can also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

As used herein, the phrase “inhibit the expression of,” refers to at an least partial “reduction” of COL1A1, TGFβ or SMAD2/3 gene expression in a cell treated with an iRNA composition as described herein compared to the expression of COL1A1, TGFβ or SMAD2/3 respectively in an untreated cell.

The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of,” and the like, in so far as they refer to a COL1A1, TGFβ and/or SMAD2/3 gene, herein refer to the at least partial suppression of the expression of the COL1A1, TGFβ and/or SMAD2/3 gene, as manifested by a reduction of the amount of COL1A1, TGFβ and/or SMAD2/3 mRNA respectively which can be isolated from or detected in a first cell or group of cells in which the COL1A1, TGFβ and/or SMAD2/3 gene is transcribed and which has or have been treated such that the expression of the COL1A1, TGFβ and/or SMAD2/3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of

(mRNA in control cells)−(mRNA in treated cells)/(mRNA in control cells)·100%

Alternatively, the degree of inhibition can be given in terms of a reduction of a parameter that is functionally linked to COL1A1, TGFβ or SMAD2/3 gene expression, e.g., the amount of protein encoded by the COL1A1, TGFβ or SMAD2/3 gene respectively, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production. In principle, COL1A1, TGFβ and/or SMAD2/3 gene silencing can be determined in any cell expressing COL1A1, TGFβ and/or SMAD2/3 either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given iRNA inhibits the expression of the COL1A1, TGFβ and/or SMAD2/3 gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.

For example, in certain instances, expression of the COL1A1, TGFβ and/or SMAD2/3 gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA featured herein. In some embodiments, the COL1A1, TGFβ and/or SMAD2/3 gene is suppressed by at least about 60%, 70%, or 80% by administration of an iRNA featured herein. In some embodiments, the COL1A1, TGFβ and/or SMAD2/3 gene is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of an iRNA as described herein.

As used herein in the context of COL1A1, TGFβ and/or SMAD2/3 expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of liver fibrosis mediated by COL1A1 overexpression. In some embodiment, the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with liver fibrosis, or to slow or reverse the fibrotic process.

By “lower” in the context of a liver function, or liver fibrosis biomarker or a liver fibrosis symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without liver failure, a chronic liver disease, liver fibrosis, or cirrhosis.

As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of liver fibrosis, which is mediated by COL1A1 overexpression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and can vary depending on factors known in the art, such as, for example, the patient's history and age, the stage of liver disease or liver fibrosis, and the administration of other agents that indirectly inhibit the COL1A1 overexpression and other agents that treat the underlying causes leading to chronic liver injury and fibrosis.

As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an iRNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting a COL1A1 gene can reduce COL1A1 protein levels by at least 10%.

The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents can include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets can be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.

As used herein, a therapeutic that “prevents” a fibrosis is a composition that, in a statistical sample, reduces the occurrence of fibrosis in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

An “infection” as used herein refers to a disease or condition attributable to the presence in a host of a foreign organism or agent that reproduces within the host. Infections typically involve breach of a normal mucosal or other tissue barrier by an infectious organism or agent. A subject that has an infection is a subject having objectively measurable infectious organisms or agents present in the subject's body.

As used herein, a “subject” is a mammal, e. g. a dog, horse, cat, and other non-human primates. In a preferred embodiment, a subject is a human.

As used herein, the term “LNPXX”, wherein the “XX” are numerals, is also referred to as “AFXX” herein. For example, LNP09 is also referred to AF09 and LNP12 is also known as or referred to as AF12.

As used herein, the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

As used herein, the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

Ii. Double-Stranded Ribonucleic Acid (dsRNA)

Described herein are iRNA agents that inhibit the expression of the COL1A1, TGFβ and/or SMAD2/3 genes. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a COL1A1, TGFβ and/or SMAD2/3 gene respectively in a cell or mammal, e.g., in a human having a liver disease characterized by hepatic fibrosis, where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a COL1A1, TGFβ or SMAD2/3 gene, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing the COL1A1, TGFβ or SMAD2/3 gene respectively, inhibits the expression of the COL1A1, TGFβ and/or SMAD2/3 gene respectively by at least 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of a COL1A1 gene in cell culture, such as in COS cells, HeLa cells, primary hepatocytes, HepG2 cells, primary cultured HSCs or in a biological sample from a subject can be assayed by measuring COL1A1 mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a COL1A1, TGFβ or SMAD2/3 gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 bp can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 bp. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex of e.g., 15-30 bps that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 bp is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, then, an miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target COL1A1, TGFβ and/or SMAD2/3 expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

In one embodiment, a COL1A1 gene is a human COL1A1 gene. In another embodiment the COL1A1 gene is a mouse or a rat COL1A1 gene. In one embodiment, an iRNA cross react with human and/or mouse and rat COL1A1 sequence. In specific embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence from Table 3 and Table 6, and the second sequence is selected from the group consisting of the corresponding antisense sequences of Table 3 and Table 6. Alternative dsRNA agents that target elsewhere in the target sequence provided in Table 3 and Table 6 can readily be determined using the target sequence and the flanking COL1A1 sequence.

In one embodiment, a TGFβ gene is a human TGFβ gene. In another embodiment the TGFβ gene is a mouse or a rat TGFβ gene. In specific embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence from Table 4 and Table 7, and the second sequence is selected from the group consisting of the corresponding antisense sequences of Table 4 and Table 7. Alternative dsRNA agents that target elsewhere in the target sequence provided in Table 4 and Table 7 can readily be determined using the target sequence and the flanking TGFβ sequence. In one embodiment, an iRNA cross react with human and/or mouse and rat TGFβ sequence.

In one embodiment, a SMAD2/3 gene is a human SMAD2/3 gene. In another embodiment the SMAD2/3 gene is a mouse or a rat SMAD2/3 gene. In specific embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence from Table 5, and the second sequence is selected from the group consisting of the corresponding antisense sequences of Table 5. Alternative dsRNA agents that target elsewhere in the target sequence provided in Table 5 can readily be determined using the target sequence and the flanking SMAD2/3 sequence. In one embodiment, an iRNA cross react with human and/or mouse and rat SMAD2/3 sequence.

In one aspect, a dsRNA will include at least two nucleotide sequences, a sense and an anti-sense sequence, whereby the sense strand is selected from the groups of sequences provided in Tables 3-7, and the corresponding antisense strand of the sense strand selected from Table 3-7. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a COL1A1, TGFβ and/or SMAD2/3 gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 3-7, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand from Tables 3-7. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences identified by sequence identifiers provided in Tables 3-7, dsRNAs described herein can include at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 3-7 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 3-7, and differing in their ability to inhibit the expression of a COL1A1, TGFβ and/or SMAD2/3 gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.

In addition, the RNAs provided in Tables 3-7 identify a site in a COL1A1, TGFβ and/or SMAD2/3 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided in Tables 3-7 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a COL1A1, TGFβ and/or SMAD2/3 gene respectively.

While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, by sequence identifier in Tables 3-7 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified by sequence identifier, e.g., in Tables 3-7, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of a COL1A1, TGFβ and/or SMAD2/3 gene, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a COL1A1, TGFβ and/or SMAD2/3 gene respectively. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of a COL1A1, TGFβ and/or SMAD2/3 gene is important, especially if the particular region of complementarity in a COL1A1, TGFβ and/or SMAD2/3 gene is known to have polymorphic sequence variation within the population.

In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39464, each of which is herein incorporated by reference

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other embodiments, suitable RNA mimetics suitable are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂-4 known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.

Another modification of the RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic stellate cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or TGFβ receptor.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic (PK) modulator. As used herein, a “PK modulator” refers to a pharmacokinetic modulator. PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as HSC in the liver. Also included are HSA and low density lipoprotein (LDL).

Cell Permeation Peptide and Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 168). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 169)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 170)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 171)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can be used, e.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an αvβ3 integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the αvβ3 integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogenesis.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

Carbohydrate Conjugates

In some embodiments, the iRNA oligonucleotides described herein further comprise carbohydrate conjugates. The carbohydrate conjugates are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4-9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C₅ and above (preferably C₅-C₈) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (preferably C₅-C₈).

In one embodiment, the carbohydrate conjugate is selected from the group consisting of:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises other ligand such as, but not limited to, PK modulator, endosomolytic ligand, and cell permeation peptide.

Linkers

In some embodiments, the conjugates described herein can be attached to the iRNA oligonucleotide with various linkers that can be cleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR⁸, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R⁸), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R⁸ is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between 1-24 atoms, preferably 4-24 atoms, preferably 6-18 atoms, more preferably 8-18 atoms, and most preferably 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, liver targeting ligands can be linked to the cationic lipids through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a preferred embodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHR^(A)C(O)NHCHR^(B)C(O)— (SEQ ID NO: 876), where R^(A) and R^(B) are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

Representative carbohydrate conjugates with linkers include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

Delivery of iRNA

The delivery of an iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

Direct Delivery

In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian R L., 1992, Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Patent No. 7, 427, 605, which is herein incorporated by reference in its entirety.

Vector Encoded dsRNAs

In another aspect, iRNA targeting the COL1A1, TGFβ and/or SMAD2/3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG., 1996, 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA, 1995, 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et at (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

III. Pharmaceutical Compositions Containing iRNA

In one embodiment, provided herein are pharmaceutical compositions containing an iRNA and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the iRNA is useful for treating hepatic fibrosis related to the overexpression a COL1A1 gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of COL1A1, TGFβ and/or SMAD2/3 genes. In general, a suitable dose of iRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The pharmaceutical composition can be administered once daily or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose on COL1A1, TGFβ and/or SMAD2/3 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

A number of mouse models for the study of liver fibrosis of various causes are available, for example, as carbon tetrachloride (CCl₄), diamethylnitrosamine and thioacetamide-treated mouse models. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose.

The present invention also includes pharmaceutical compositions and formulations that include the iRNA compounds featured in the invention. The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the HSCs of the liver).

Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent can act.

Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al, Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al, Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al, disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1, 2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C_(1215G), that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describes PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes can include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). Nucleic acid lipid particles

In one embodiment, a COL1A1, TGFβ and SMAD2/3 dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-42-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98.4 HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are as follows:

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Cationic Lipid Lipid:siRNA ratio SNALP-1 1,2-Dilinoleyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG- dimethylaminopropane (DLinDMA) cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~ 7:1 S-XTC 2,2-Dilinoleyl-4- XTC/DPPC/Cholesterol/PEG- dimethylaminoethyl-[1,3]-dioxolane cDMA (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~ 7:1 LNP05 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl-[1,3]-dioxolane 57.5/7.5/31.5/3.5 (XTC) lipid:siRNA ~ 6:1 LNP06 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl-[1,3]-dioxolane 57.5/7.5/31.5/3.5 (XTC) lipid:siRNA ~ 11:1 LNP07 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl-[1,3]-dioxolane 60/7.5/31/1.5, (XTC) lipid:siRNA ~ 6:1 LNP08 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl-[1,3]-dioxolane 60/7.5/31/1.5, (XTC) lipid:siRNA ~ 11:1 LNP09 2,2-Dilinoleyl-4- XTC/DSPC/Cholesterol/PEG-DMG dimethylaminoethyl-[1,3]-dioxolane 50/10/38.5/1.5 (XTC) Lipid siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG- di((9Z,12Z)-octadeca-9,12- DMG dienyl)tetrahydro-3aH- 50/10/38.5/1.5 cyclopenta[d][1,3]dioxol-5-amine Lipid:siRNA 10:1 (ALN100) LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG 6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- C12-200/DSPC/Cholesterol/PEG- hydroxydodecyl)amino)ethyl)(2- DMG hydroxydodecyl)amino)ethyl)piperazin- 50/10/38.5/1.5 1-yl)ethylazanediyl)didodecan- Lipid:siRNA 10:1 2-ol (C12-200) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG- DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.

XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference.

ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y), wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)R^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dime thylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above can be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R₃ and R₄ are independently lower alkyl or R₃ and R₄ can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO₃solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H]− 232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO₃(1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]−266.3, [M+NH₄+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR □=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

Additional Formulations Emulsions

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials is also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (P0500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts. The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agent. Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293 Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more other agents which function by a non-RNAi mechanism. Examples of such other agents include but are not limited one or more recombinant cytokines (e.g., IL6, IFN-γ, and TNF), PPAR gamma ligands, and corticosteroids. Basically, the other non-RNAi agents are targeted at the underlying causes of the liver fibrosis and/or aimed at alleviating the symptoms of the condition. Therapeutics for the underlying causes of liver fibrosis are known in the art. Some of such therapeutics and strategies are shown in Table 2.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein lies generally within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured herein can be administered in combination with other known agents effective in treatment of hepatic fibrosis mediated by COL1A1 overexpression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

IV. The iRNA Target Genes: COL1A1, TGFβ, and SMAD2/3

The Homo sapiens collagen, type I, alpha 1 (COL1A1) is also known as collagen alpha-1(I) chain; alpha-1 type I collagen; pro-alpha-1 collagen type 1; collagen alpha 1 chain type I; collagen of skin, tendon and bone, alpha-1 chain; 014; and COL1A1.

The COL1A1 gene encodes the pro-alpha1 chains of type I collagen whose triple helix comprises two alpha1 chains and one alpha2 chain. Type I collagen is a fibril-forming collagen found in most connective tissues and is abundant in bone, cornea, dermis and tendon. Mutations in this gene are associated with osteogenesis imperfecta types I-IV, Ehlers-Danlos syndrome type VIIA, Ehlers-Danlos syndrome Classical type, Caffey Disease and idiopathic osteoporosis. Reciprocal translocations between chromosomes 17 and 22, where this gene and the gene for platelet-derived growth factor beta are located, are associated with a particular type of skin tumor called dermatofibrosarcoma protuberans, resulting from unregulated expression of the growth factor. Two transcripts, resulting from the use of alternate polyadenylation signals, have been identified for this gene. The human COL1A1 gene is conserved in chimpanzee, dog, cow, mouse, rat, and zebrafish.

The GENBANK™ Accession No. for the human COL1A1 mRNA is NM_000088.3 and the sequence is provided herein as SEQ ID NO: 172.

The GENBANK™ Accession No. for the mouse COL1A1 mRNA is NM_007742.3 and the sequence is is provided herein as SEQ ID NO: 173.

The Homo sapiens transforming growth factor, beta 1 (TGFβ1) is also known as TGF-beta-1, TGF-beta 1 protein, latency-associated peptide, CED, LAP, DPD1, TGFb and TGFbeta. This gene encodes a member of the transforming growth factor beta (TGFβ) family of cytokines, which are multifunctional peptides that regulate proliferation, differentiation, adhesion, migration, and other functions in many cell types. Many cells have TGFβ receptors, and the protein positively and negatively regulates many other growth factors. The secreted protein is cleaved into a latency-associated peptide (LAP) and a mature TGFβ1 peptide, and is found in either a latent form composed of a TGFβ1 homodimer, a LAP homodimer, and a latent TGFβ1-binding protein, or in an active form composed of a TGFβ1 homodimer. The mature peptide can also form heterodimers with other TGFβ family members. The human TGFβ1 gene is conserved in chimpanzee, dog, mouse, rat, and zebrafish.

The GENBANK™ Accession No. for the human TGFβ1 mRNA is 1.NM_000660.4 and the sequence is provided herein as SEQ ID NO: 174.

The Genbank™ Accession No. for the mouse TGFβ1 mRNA is 1.NM_011577.1; GI:6755774 and the sequence is provided herein as SEQ ID NO: 175.

The Homo sapiens SMAD family member 2 (SMAD2) is also known as “mothers against decapentaplegic homolog 2”, MAD homolog 2, Mad protein homolog, Mad-related protein 2, mother against DPP homolog 2, mothers against DPP homolog 2, Sma- and Mad-related protein 2, JV18; MADH2; MADR2; JV18-1; hMAD-2; hSMAD2; MGC22139; MGC34440; and SMAD2. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. SMAD2 is recruited to the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation (SARA) protein. In response to TGF-beta signal, SMAD2 is phosphorylated by the TGF-beta receptors. The phosphorylation induces the dissociation of this protein with SARA and the association with the family member SMAD4. The association with SMAD4 is important for the translocation of this protein into the nucleus, where it binds to target promoters and forms a transcription repressor complex with other cofactors. SMAD2 can also be phosphorylated by activin type 1 receptor kinase, and mediates the signal from the activin. Alternatively spliced transcript variants encoding the same protein have been observed. The human SMAD2 gene is conserved in chimpanzee, dog, cow, mouse, rat, chicken, and zebrafish.

There are three known isoforms of the human SMAD2 transcipts. The GENBANK™ Accession Nos. for these three human SMAD2 mRNAs are NM_001003652.2, NM_001135937.1 and NM_005901.4 and the sequences are provided herein as SEQ ID NO: 176, SEQ ID NO: 177, and SEQ ID NO: 178, respectively.

There is one known transcript for the mouse SMAD2. The GENBANK™ Accession Nos. for the mouse SMAD2 mRNAs is NM_010754.4 and the sequence is provided herein as SEQ ID NO: 179.

The Homo sapiens SMAD family member 3 (SMAD3) is also known as “mothers against decapentaplegic homolog 3”, MADH3, hMAD-3, hSMAD3, MAD homolog 3, mad protein homolog, mothers against DPP homolog 3, SMA- and MAD-related protein 3, mad homolog JV15-2, HSPC193, HsT17436, MGC60396, DKFZp586N0721, and DKFZp686J10186.

The protein encoded by this gene belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene ‘mothers against decapentaplegic’ (Mad) and the C. elegans gene Sma. SMAD3 mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. SMAD3 functions as a transcriptional modulator activated by transforming growth factor-beta and is thought to play a role in the regulation of carcinogenesis. The human SMAD3 gene is conserved in chimpanzee, dog, cow, mouse, rat, chicken, zebrafish, fruit fly, and mosquito.

There are at least four isoforms of the SMAD3 transcipts. The GENBANK™ Accession Nos. for the four human SMAD3 mRNAs are NM_001145102.1, NM_001145103.1, NM_001145104.1, and NM_005902.3 and the sequences are provided herein as SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, and SEQ ID NO: 183, respectively.

There is one known transcript for the mouse SMAD3. The GENBANK™ Accession Nos. for the mouse SMAD3 mRNAs is NM_016769.4 and the sequence is provided herein as SEQ ID NO: 184.

V. The Liver, Liver Disease, Fibrobsis and Cirrhosis

Fibrosis is characterized by the formation of an abnormal amount of fibrous connective tissue in an organ or part as the result of inflammation, irritation, or healing. Fibrotic disorders, include, but are not limited to, systemic sclerosis (SSc), idiopathic pulmonary fibrosis (IPF), interstitial lung disease (ILD), cirrhosis of the liver, nephrogenic systemic fibrosis (NSF), pneumoconiosis (chronic respiratory disease caused by inhaling metallic or mineral particles), fasciitis, scleromyxedema, retroperitoneal fibrosis, pulmonary fibrosis, liver fibrosis, chronic graft versus host disease and chronic allograft rejection and cystic fibrosis, are characterized by abnormal and excessive deposition of collagen and other extracellular matrix (ECM) components in various tissues. Active fibrosis also can occur in rheumatoid arthritis, lupus, autoimmune diseases, Lyme disease, asthma, idiopathic pulmonary fibrosis, chronic pulmonary fibrosis, uterine fibrosis, and ovarian fibrosis. Although their etiology is quite diverse, the presence of ECM-producing fibroblasts displaying an activated phenotype in the affected tissues is typical of all fibrotic diseases. Fibroblast activation is characterized by a marked increase in the transcriptional activity of the genes encoding type I and type III collagens and fibronectin, initiation of the expression of alpha-smooth muscle actin (α-SMA), and the reduction of ECM degradative activities. Activated fibroblasts display contractile properties resulting from the expression of stress fibers containing α-SMA, and their pro-fibrotic activation is part of a complex set of molecular and biochemical changes that are conserved for multiple sequential passages in vitro.

In the body, the liver is the most complex and metabolically active organ and it performs over 500 vital functions. It is also the major detoxification organ in the body. The liver regulates most chemical levels in the blood and secretes bile, which helps carry away waste products from the liver. All the blood leaving the stomach and intestines passes through the liver. The liver processes this blood and breaks down the nutrients and other components into forms that are become available for use for the rest of the body. Some of the important functions are as follows: (1) provides immunity against infection; (2) manufactures most of the important proteins in the body, and also cholesterol and lipoproteins in which all body fats are carried; (3) clears the blood of most chemicals, drugs and alcohol; (4) excretes bile into the intestine. Bile is vital for digestion of fats, and also serves to carry body wastes; (5) regulates clotting of blood by manufacturing vital proteins; and (6) converts and stores extra sugar (glucose) in the form of starch (glycogen) which can be used in times of starvation. In addition, the liver has an enormous reserve function capacity. All liver functions remain normal even if 70% of it is removed. The liver is also the only organ in the body which can regenerate itself after large portions of it are removed.

Fibrosis of the liver occurs when an abnormal amount of fibrous connective tissue is produced as the result of inflammation, irritation, or healing caused by an underlying acute or chronic liver condition. Hepatitis is an inflammation of the liver that results in liver cell damage and destruction. Hepatitis can be acute, i.e., acute liver disease, or chronic, i.e., chronic liver disease. Patients who do not recover fully from acute hepatitis can develop chronic hepatitis, as the liver continues to sustain more damage and inflammation. Hepatitis is considered chronic if symptoms persist longer than three months to six months. Inflammation/irritation/healing in a chronic liver condition can be due to a viral (e.g. hepatitis A, B, C, D, E or G) or other infection (Entamoeba histolytica, malaria parasite Plasmodium sp., roundworm Trichinella spiralis, the Chinese liver fluke, Clonorchis sinensis, and fluke of the genus Schistosoma); autoimmune hepatitis; bile duct obstruction or billiary diseases due to biliary atresia, a congenital disorder, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC); metabolic disorders (e.g., hemochromatosis—too much iron; Wilson's Disease—too much copper), fat accumulation due to alcohol abuse or the idiopathic non-NASH, which is often associated with diabetes and obesity; and exposure to toxic chemicals, e.g., overdose of drugs such as acetaminophen, paracetamol, dry cleaning chemicals, and some wild mushrooms; and liver cancer. Approximately 90 percent of patients who develop liver tumors suffer from cirrhosis. Types of liver cancer include: hepatocellular carcinoma, fibrolamellar carcinoma, secondary liver cancer, and cholangiocarcinoma (bile duct tumor). Other causes of liver fibrosis include obstruction of outflow of blood from the liver (i.e., Budd-Chiari syndrome), Crigler-Najjar syndrome, Gilbert syndrome, Dubin-Johnson syndrome, hyperbilirubinemia, heart and blood vessel disturbances, alpha1-antitrypsin deficiency, high blood galactose levels, high blood tyrosine levels at birth, glycogen storage disease, diabetes and malnutrition.

The process that initiates the progression towards chronic liver disease and fibrosis of the liver starts with an acute hepatitis. If acute hepatitis is not resolved, chronic hepatitis occurs and sets the stage for chronic liver disease characterized by fibrosis, and in some cases, cirrhosis. As an illustration, alcohol-induced liver disease caused by excessive consumption of alcohol starts with the fatty liver (Stage I). Fatty liver is excessive accumulation of fat inside the liver cells. This is the most common alcohol-induced liver disorder. The liver is enlarged, causing upper abdominal discomfort on the right side. In stage II, alcoholic hepatitis occurs which is an acute inflammation of the liver, accompanied by the destruction of individual liver cells and scarring. Symptoms can include fever, jaundice, an increased white blood cell count, an enlarged, tender liver, and spider-like veins in the skin. Stage III is alcoholic cirrhosis, characterized by the destruction of normal liver tissue, leaving non-functioning scar tissue. Symptoms can include those of alcoholic hepatitis, in addition to portal hypertension (leading to blood vomiting), enlarged spleen, ascites, excessive bleeding (due to poor clotting), kidney failure, confusion, or liver cancer.

Cirrhosis of the liver is a chronic disease of the liver characterized by the replacement of normal tissue with fibrous tissue and the loss of functional liver cells. The general architecture of liver cirrhosis is regenerative nodules surrounded by dense fibrotic tissue. The overgrowth of fibrosis scar tissue inhibits the liver's proper functioning. Liver fibrosis usually results in cirrhosis and is a late stage of hepatic fibrosis that has resulted in widespread distortion of normal hepatic architecture. The causes of cirrhosis are the same as those of fibrosis. Cirrhosis is usually considered irreversible. This irreversible scarring of the liver can be life-threatening. In an advanced stage, 80-90% liver can be damaged and replaced with scar tissue. Symptoms of cirrhosis vary, depending on severity. Mild cirrhosis can not exhibit any symptoms at all. Initial symptoms of liver cirrhosis can include general symptoms such as tiredness, lethargy, yellowness of eyes and urine (mild jaundice), swollen feet, excessive itching and anemia (low hemoglobin). In more advanced stages, the patient can have severe life threatening complications such as blood vomiting, bloated stomach due to water (ascites) in the abdomen which can develop serious infection, mental deterioration and coma, deep jaundice and kidney impairment. In addition, the patient can have a bleeding tendency due to low levels of the liver protein prothrombin, and low platelet count both of which are vital for normal clotting of blood.

Fibrosis and cirrhosis of the liver can be assessed and diagnosed by medical procedures known to one skilled in the art. The medical procedures include but are not limited to specific laboratory tests, liver function tests, liver biopsy, Doppler ultrasonography, CT and/or MR imaging and cholangiography (x-rays of the bile ducts).

Specific laboratory tests, liver and function tests include non-invasive diagnostic procedures comprising a series of blood tests which can often determine whether or not the liver is functioning properly. These tests can also distinguish between acute and chronic liver disorders and between hepatitis and cholestasis. The most commonly performed blood tests include: (1) serum bilirubin test—elevated levels of bilirubin often indicate an obstruction of bile flow or a defect in the processing of bile by the liver-bilirubin is produced by the liver and is excreted in the bile; (2) serum albumin test—below-normal levels of albumin, are associated with many chronic liver disorders; (3) serum alkaline phosphatase test-elevated levels of alkaline phosphatase, an enzyme found in the bile, usually indicate an obstruction of bile flow, liver injury, or certain cancers; (4) serum aminotransferases (transaminases)—this enzyme is released from damaged liver cells; (5) prothrombin time (PTT) test—this test measures the time it takes for blood to clot. Blood clotting requires vitamin K and a protein made by the liver. Liver cell damage and bile flow obstruction can both interfere with proper blood clotting; (7) alanine transaminase (ALT) test—this enzyme is released from damaged liver cells; (8) aspartate transaminase (AST) test—this enzyme is released from damaged liver, heart, muscle, or brain cells; (9) gamma-glutamyl transpeptidase test—this enzyme is produced by the liver, pancreas, and kidneys and released into the blood when these organs are injured; (10) lactic dehydrogenase test—this enzyme is released when organs such as the liver, heart, lung, or brain are injured; (11) 5-nucleotidase test—this enzyme is released by the liver when the liver is injured due to bile duct obstruction or impaired bile flow; (12) alpha-fetoprotein test—this protein is produced by the fetal liver and testes, indicating hepatitis or cancer when hepatisits is experienced in adults; (13) mitochondrial antibodies test—the presence of these antibodies can indicate primary biliary cirrhosis, chronic active hepatitis, and certain other autoimmune disorders. In some embodiments, biomarkers of fibrosis are used to assess and diagnose liver fibrosis and/or cirrhosis. Biomarkers of liver fibrosis are known in the art, for example, see Table 9. Test results may be normal or may detect nonspecific abnormalities due to complications of cirrhosis or alcoholism. ALT and AST levels are often modestly elevated. Alkaline phosphatase and γ-glutamyl transpeptidase (GGT) are often normal; elevated levels indicate cholestasis or biliary obstruction. Bilirubin is usually normal but increases when cirrhosis progresses, particularly in primary biliary cirrhosis (see below). Decreased serum albumin and a prolonged PT directly reflect impaired hepatic synthesis—usually an end-stage event. Albumin can also be low when nutrition is poor. Serum globulin increases in cirrhosis and in most liver disorders with an inflammatory component. Anemia is common and usually normocytic with a high RBC distribution width. Anemia is often multifactorial: microcytic from chronic GI bleeding; macrocytic from folate nutritional deficiency or hemolysis (especially in alcohol abuse) and hypersplenism. CBC can also detect leucopenia, thrombocytopenia, or pancytopenia.

Liver biopsy is currently considered the gold standard for the accurate diagnosis of liver fibrosis and cirrhosis, or for ruling out any coexisting liver disease, staging and grading the severity of liver disease, treatment decisions, patient and provider reassurance, and as a benchmark for gauging future disease progression. A diagnosis biopsy is a very important and helpful test in the diagnosis of numerous diseases that affect the liver and bile ducts. In most cases, this allows establishment of a very specific diagnosis and also permit staging and grading of the condition. Several staging and grading scales are known to in the art, for example, see Table 8 (F. Grünhage and F. Lammert, Chapter 20: Assessment of hepatic fibrosis in chronic viral hepatitis, in Hepatology: a clinical textbook. Eds. Mauss, Berg, Rockstroh, Sarrazin and Wedemeyer, 2nd ed. 2010) and the Child-Turcotte-Pugh Scoring System (Merck Medical Manuals). A monitoring liver biopsy can help the doctor monitor the effectiveness of therapy that the patient is receiving for a disease that affects the liver. There are many different types of liver biopsies, such as the percutaneous (via a needle through the skin), transjugular (through the blood vessels), laparoscopic (via small abdomen incisions), fine needle aspiration and open surgery liver biopsy. During the procedure, small pieces of liver tissue are removed in order to be sent to a laboratory for histological examination.

Other non-invasive techniques for diagnosis and staging liver fibrosis are also used. For example, liver fibrosis markers: α-2-macroglobulin(α-MA), transferrin, apolipoproteinA 1, hyaluronic acid (HA), laminin, N-terminal procollagen III(PIIINP), 7S collagen IV (7S-IV), total bilirubin, indirect bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase(AST), AST/ALT, g-glutamyl transpeptidase(GGT), alkaline phosphatase(ALP), albumin, albumin/globulin, blood urea nitrogen(BUN), creatinine(Cr), triglyceride, cholersterol, and high density lipoprotein and low density lipoprotein. Lun-Gen Lu et al. (World J. Gastroenterologgy, 2003, 9(11):2574-2578) measured over 20 non-invasive parameters of 200 patients with chronic liver disease and attempted to correlate inflammatory activity, as determined by the levels of the non-invasive parameters, with fibrosis of consecutive liver biopsies from these patients in order to stage and grade liver fibrosis without having to perform the gold standard liver biopsy. In pathological diagnosis, Stage 1 and Stage 2 indicated mild fibrosis, Stage 3 and Stage 4 indicated severe fibrosis. Lun-Gen Lu et al. found that there was a close correlation between liver fibrosis and inflammatory activity. AST, GGT, albumin, albumin/globulin, ALP, AFP, hyaluronic acid, N-terminal procollagen III(P III NP), collagen type IV(Col IV), tissue inhibitors of metalloproteinases-1(TIMP-1), alpha-2-macroglobulin, natural killer cells(NK), some parameters of Doppler ultrasonography, CT and/or MR imaging were all related to the degree of inflammatory activity. GGT, albumin, albumin/globulin, ALP, AFP, hyaluronic acid, Col IV, TIMP-1, alpha-2-macroglobulin, transforming growth factor-beta 1(TGFb1), NK, some parameters of Doppler ultrasonography, CT and/or MR imaging were all related to the staging of fibrosis. It was found that the level of TGFβ and TIMP-1 increased more significantly in inflammation and early stage of fibrosis, indicating that they could reflect the changes of liver inflammation and fibrosis. GGT, albumin, albumin/globulin, ALP, AFP, HA, 7S-IV, and α2-MA had a positive correlation with liver fibrosis. In this study, PIIINP, laminin, transferrin, and apoproteinA1 which were known to have significant diagnostic value, were not confirmed.

Treatment of liver disease and consequent liver fibrosis includes, but is not limited to, treating the primary cause of the inflammation or irritation in the liver and controlling the inflammation (see Table 2). In addition to the dsRNA-mediated approaches described herein, treatment can also include one/more of the following: Antiviral Agents—when liver disease or liver fibrosis is caused by hepatitis B or C, inflammation of the liver can be stopped with the injectable antiviral drug interferon-alpha. In addition, in Hepatitis B, oral anti-viral agents such as lamivudine or adefovir can be used, while in Hepatitis C, ribavirin can be used. Corticosteroids—corticosteroids can be used to treat chronic liver disease caused by an autoimmune disorder. However, even when inflammation is suppressed, it is possible that scarring of the liver may continue. Discontinuation of certain drugs—when chronic hepatitis is caused by certain drugs, discontinuing those drugs usually clears up any symptoms. Discontinuance of alcohol—while this is essential for recovery in alcohol induced chronic liver disease, it is also highly advisable in Hepatitis C and other chronic diseases of the liver. Examples of other therapies include those disclosed in Table 2. In addition, traditional Chinese medicine such as Qinggan Capsule, ginkgo leaf, Ruangan, salvia, bupleurum and white peony can be used in combination with the therapeutic methods and compositions for the treatment, prevention and/or management of liver fibrosis.

VI. Methods for Inhibiting Expression of a COL1A1, TGFβ, and/or SMAD2/3 Gene

In one aspect, provided herein is a method of inhibiting COL1A1 expression in a cell, the method comprising: (a) introducing into the cell a dsRNA that targets a COL1A1, TGFβ or SMAD2/3 gene in the cell; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a COL1A1, TGFβ or SMAD2/3 gene respectively, thereby inhibiting expression of the COL1A1 gene in the cell. In the embodiments where the dsRNA targets TGFβ and/or SMAD2/3 gene expression, the expression of the COL1A1 gene in the cell is indirectly inhibited by way of reduced signaling from TGFβ which stimulates the cell to overexpress COL1A1. The inhibition is not by way of degradation of the mRNA transcript of the COL1A1 gene. Reduction in gene expression can be assessed by any methods known it the art and by methods, e. g. qRT-PCR, described herein.

In one embodiment, the cell is a mammalian cell, preferably a human cell. In another embodiment, the cell is a mammalian liver cell. In a preferred embodiment, the cell is a HSC.

In one embodiment, more than one dsRNA is introduced into the cell. In one embodiment, the dsRNAs targeting COL1A1, TGFβ and/or SMAD2/3 gene are introduced to the cell, i.e. a combination of dsRNA are introduced into the cell and more that one gene is targeted. For example, a combination of COL1A1iRNA and TGFβiRNA, a combination of COL1A1iRNA and SMAD2/3iRNA or a combination of COL1A1iRNA, TGFβiRNA and SMAD2/3iRNA.

In one embodiment, the dsRNA is introduced to the cell, preferably, in a liposome, e.g. a LNP-formulated liposome known in the art and/or described herein. In one embodiment, the LNP is formulated to target a specific cell such as a HSC. For example, the LNP-formulated liposome can be coupled to Vitamin A in order to target the LNP-encapsulated dsRNA to the vitamin A-sequestering HSC.

In one embodiment, the COL1A1 expression is inhibited by at least, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100%. In the embodiments where TGFβ and/or SMAD2/3 genes expression are also inhibited by dsRNAs, their respectively expressions are inhibited by at least 30%, in addition to a decrease in COL1A1 expression.

In one aspect, provided herein is a method for inhibiting the expression of a COL1A1 gene in a mammal, the method comprising (a) administering to the mammal a composition comprising a dsRNA that targets a COL1A1, TGFβ, or SMAD2/3 gene in a cell of the mammal; and (b) maintaining the mammal of step (a) for a time sufficient to obtain degradation of the mRNA transcript of a COL1A1, TGFβ, or SMAD2/3 gene respectively, thereby inhibiting expression of the COL1A1 gene in the cell. In the embodiments where the dsRNA targets TGFβ and/or SMAD2/3 gene expression, the expression of COL1A1 in the cell is indirectly inhibited by way of reduced signaling from TGFβ which stimulates the cell to overexpress COL1A1; the inhibition is not by way of degradation of the mRNA transcript of the COL1A1 gene. Reduction in gene expression can be assessed by any methods known it the art and by methods, e. g. qRT-PCR, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the target gene expression.

In one embodiment, the method includes administering a composition featured herein to the mammal such that expression of the target COL1A1 gene is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target COL1A1, TGFβ, or SMAD2/3 gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described elsewhere herein.

In some embodiments of the aspects described herein, the methods and compositions featured herein comprise more than one iRNA such that more than one iRNA is administered to the mammal and more than one gene is targeted in the mammal. For example, a combination of COL1A1iRNA and TGFβiRNA, a combination of COL1A1iRNA and SMAD2/3iRNA or a combination of COL1A1iRNA, TGFβiRNA and SMAD2/3iRNA.

Accordingly, in some embodiments of the aspects described herein, the methods and the composition described herein comprises iRNAs targeting a COL1A1gene and a TGFβ gene. In these methods, both the COL1A1 and TGFβ gene expressions are inhibited. In another embodiment, the methods and the composition described herein comprises iRNAs targeting a COL1A1gene and a SMAD2/3 gene. In these methods, both the COL1A1 and SMAD2/3 gene expressions are inhibited. In yet another embodiment, the methods and the composition described herein comprises iRNAs targeting a COL1A1gene, a TGFβ gene and a SMAD2/3 gene. In these methods, all three the COL1A1, SMAD2/3 and TGFβ gene expressions are inhibited.

In one embodiment of the aspects described herein, the method includes administering a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the COL1A1, TGFβ, or SMAD2/3 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection.

VII. Methods for Treating Liver Disease and Liver Fibrosis Characterized by the Overexpression of a COL1A1 Gene

Aspects and embodiments of the invention relate in particular to the use of an iRNA targeting COL1A1 gene expression and compositions containing at least one iRNA for the treatment of a COL1A1-mediated liver fibrosis. Embodiments of the invention also relate to the use of an iRNA targeting the molecules in the signaling pathway that promote COL1A1 gene expression, e. g. TFGβ and SMAD2/3. For example, a composition containing an iRNA targeting a COL1A1 gene is used for treating and/or preventing liver fibrosis or a composition containing an iRNA targeting a TGFβ and/or SMAD2/3 gene is used for treating and/or preventing liver fibrosis

In one embodiment, provided herein is a method of treating and/or preventing hepatic fibrosis in a subject in need of such treatment, the method comprising administering to the subject a therapeutically effective amount of the iRNA that targets a COL1A1, TGFβ, or SMAD2/3 gene.

In one embodiment, the method of treating and/or preventing hepatic fibrosis comprises (a) administering to the subject a composition comprising a iRNA that targets a COL1A1, TGFβ, or SMAD2/3 gene in a cell of the mammal; and (b) maintaining the subject of step (a) for a time sufficient to obtain degradation of the mRNA transcript of a COL1A1, TGFβ, or SMAD2/3 gene respectively, thereby inhibiting or reducing expression of the COL1A1 gene in the cell. In the embodiments where the iRNA targets TGFβ and/or SMAD2/3 gene expression, the expression of COL1A1 in the cell is indirectly reduced by way of reduced signaling from TGFβ which stimulates the cell to over express COL1A1; the reduction of COL1A1 expression is not by way of degradation of the mRNA transcript of the COL1A1 gene. Reduction in gene expression can be assessed by any methods known it the art and by methods, e. g. qRT-PCR, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the target gene expression.

In one embodiment, the liver fibrosis is a result of a viral or other infection, an autoimmune disorder, a bile duct obstruction, metabolic disorders, alcoholic abuse, primary biliary cirrhosis, NASH, exposure to chemicals or cancer. In other embodiments, the liver fibrosis is the results of other indications described herein.

In one embodiment, more than one dsRNA is administered to the subject, and as a result thereof, more than one gene is targeted. For example, a combination of COL1A1 iRNA and TGFβ iRNA, a combination of COL1A1 iRNA and SMAD2/3 iRNA or a combination of COL1A1 iRNA, TGFβ iRNA and SMAD2/3 iRNA.

In one embodiment, the method of treating and/or preventing hepatic fibrosis described herein comprises iRNAs targeting a COL1A1gene and a TGFβ gene. In this embodiment, both the COL1A1 and TGFβ gene expressions are inhibited. In another embodiment, the method of treating and/or preventing hepatic fibrosis described herein comprises iRNAs targeting a COL1A1gene and a SMAD2/3 gene. In this embodiment, both the COL1A1 and SMAD2/3 gene expression are inhibited. In yet another embodiment, the method of treating and/or preventing hepatic fibrosis described herein comprises iRNAs targeting a COL1A1gene, a TGFβ gene and a SMAD2/3 gene. In this embodiment, all three of the COL1A1, SMAD2/3 and TGFβ gene expressions are inhibited.

In one embodiment, iRNAs targeting a COL1A1gene, a TGFβ gene, and a SMAD2/3 gene are administered.

In one embodiment, the iRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.

In one embodiment, the method further relates to the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating and/or preventing chronic liver disease or hepatic fibrosis, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, for example, those which are currently employed for treating the underlying causes of the chronic liver disease or hepatic fibrosis. For example, the iRNA or pharmaceutical composition thereof can also be administered in conjunction with one or more additional anti-hepatic cancer treatments, such as biological chemotherapy and radiation. Accordingly, a treatment can include, for example, chemotherapy (for example, chlorambucil, prednisone, prednisolone, vincristine, cytarabine, clofarabine, farnesyl transferase inhibitors, decitabine, inhibitors of MDR1), rituximab, interferon-α, anthracycline drugs (such as daunorubicin or idarubicin), L-asparaginase, doxorubicin, cyclophosphamide, doxorubicin, bleomycin, fludarabine, etoposide, pentostatin, or cladribine), bone marrow transplant, stem cell transplant, anti-metabolite drugs (methotrexate and 6-mercaptopurine), or any combination thereof.

Chemotherapy is the treatment of cancer with drugs that can destroy cancer cells. In current usage, the term “chemotherapy” usually refers to cytotoxic drugs which affect rapidly dividing cells in general, in contrast with targeted therapy. Chemotherapy drugs interfere with cell division in various possible ways, e.g. with the duplication of DNA or the separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly dividing cells and are not specific to cancer cells, although some degree of specificity may come from the inability of many cancer cells to repair DNA damage, while normal cells generally can. Most chemotherapy regimens are given in combination. Exemplary chemotherapeutic agents include, but are not limited to, 5-FU Enhancer, 9-AC, AG2037, AG3340, Aggrecanase Inhibitor, Aminoglutethimide, Amsacrine (m-AMSA), Asparaginase, Azacitidine, Batimastat (BB94), BAY 12-9566, BCH-4556, Bis-Naphtalimide, Busulfan, Capecitabine, Carboplatin, Carmustaine+ Polifepr Osan, cdk4/cdk2 inhibitors, Chlorombucil, CI-994, Cisplatin, Cladribine, CS-682, Cytarabine HCl, D2163, Dactinomycin, Daunorubicin HCl, DepoCyt, Dexifosamide, Docetaxel, Dolastain, Doxifluridine, Doxorubicin, DX895 if, E 7070, EGFR, Epirubicin, Erythropoietin, Estramustine phosphate sodium, Etoposide (VP16-213), Farnesyl Transferase Inhibitor, FK 317, Flavopiridol, Floxuridine, Fludarabine, Fluorouracil (5-FU), Flutamide, Fragyline, Gemcitabine, Hexamethylmelamine (HMM), Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Interleukin-2, Irinotecan, ISI 641, Krestin, Lemonal DP 2202, Leuprolide acetate (LHRH-releasing factor analogue), Levamisole, LiGLA (lithium-gamma linolenate), Lodine Seeds, Lometexol, Lomustine (CCNU), Marimistat, Mechlorethamine HCl (nitrogen mustard), Megestrol acetate, Meglamine GLA, Mercaptopurine, Mesna, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Mitotane (o.p′-DDD), Mitoxantrone, Mitoxantrone HCl, MMI 270, MMP, MTA/LY 231514, Octreotide, ODN 698, OK-432, Oral Platinum, Oral Taxoid, Paclitaxel (TAXOL®), PARP Inhibitors, PD 183805, Pentostatin (2′ deoxycoformycin), PKC 412, Plicamycin, Procarbazine HCl, PSC 833, Ralitrexed, RAS Farnesyl Transferase Inhibitor, RAS Oncogene Inhibitor, Semustine (methyl-CCNU), Streptozocin, Suramin, Tamoxifen citrate, Taxane Analog, Temozolomide, Teniposide (VM-26), Thioguanine, Thiotepa, Topotecan, Tyrosine Kinase, UFT (Tegafur/Uracil), Valrubicin, Vinblastine sulfate, Vindesine sulfate, VX-710, VX-853, YM 116, ZD 0101, ZD 0473/Anormed, ZD 1839, ZD 9331.

Efficacy of treatment of liver fibrosis or amelioration of liver fibrosis can be assessed, for example by periodic monitoring liver fibrosis markers: α-2-macroglobulin(α-MA), transferrin, apolipoproteinAl, hyaluronic acid (HA), laminin, N-terminal procollagen III(PIIINP), 7S collagen IV (7S-IV), total bilirubin, indirect bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase(AST), AST/ALT, g-glutamyl transpeptidase(GGT), alkaline phosphatase(ALP), albumin, albumin/globulin, blood urea nitrogen(BUN), creatinine(Cr), triglyceride, cholersterol, high density lipoprotein and low density lipoprotein and liver puncture biopsy. Liver fibrosis markers can be measured and/or liver puncture biopsy can be performed before treatment (initial readings) and subsequently (later readings) during the treatment regimen. Comparisons of the later readings with the initial readings provide a physician indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting COL1A1 or pharmaceutical composition thereof, “effective against” a hepatic fibrosis condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating liver fibrosis and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameter markers of hepatic fibrosis status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment, for example, a lower level of liver enzyme activity in the serum or an increase in serum albumin indicating an improvement in liver's ability to synthesize albumin. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model of liver fibrosis as known in the art, e.g. a mouse model described herein. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker, symptom or extracellular collagen as viewed histologically is observed.

Infection by viruses and parasites can cause inflammation and hepatic fibrosis. Some examples are the Hepadnaviridae (Hepatitis A and B viruses); Hepatitis D virus, Hepatitis E virus, and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=enterally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses). Exemplary parasites include, but are not limited to: Entamoeba histolytica; the malaria parasite Plasmodium species (Plasmodium falciparum, P. malariae, P. ovale, P. vivax), the nematode Trichinella spiralis, the trematods Clonorchis sinensis, Schistosoma mansoni, S. haematobium, and S. japonicum and any combination thereof.

In other embodiments, administration of an iRNA targeting COL1A1, TGFβ and/or SMAD2/3 is performed in combination with an anti-viral medicament or agent. Exemplary antiviral agents useful for the methods described herein include, but are not limited to, immunoglobulins, amantadine, interferon, nucleoside analogues, and protease inhibitors. Specific examples of antiviral agents include but are not limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride; Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine; Zidovudine; and Zinviroxime.

In further embodiments, administration of an iRNA targeting COL1A, TGFβ and/or SMAD2/3 is administered in combination with an anti-parasitic medicament or agent. An “antiparasitic medicament” refers to an agent that kills or inhibits the growth or function of infective parasites. Examples of antiparasitic medicaments, also referred to as parasiticides, useful for the methods described herein include, but are not limited to, albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, doxycycline, eflomithine, furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate), suramin, tetracycline, thiabendazole, timidazole, trimethroprim-sulfamethoxazole, and tryparsamide, some of which are used alone or in combination with others.

The iRNA and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.

In one embodiment, the iRNA is administered with a non-iRNA therapeutic agent, such as an agent known to treat the underlying cause of the liver disease or hepatic fibrosis, e.g., a viral or other infection, an autoimmune disorder, a bile duct obstruction, metabolic disorders, alcohol abuse, primary biliary cirrhosis, NASH, exposure to chemicals, and liver cancer. Some examples of non-iRNA therapeutics are shown in Table 2. In another embodiment, the iRNA featured described herein, e.g., a dsRNA targeting COL1A1, TGFβ and/or SMAD2/3, is administered along with a non-iRNA therapeutic agent, such as an anti-hepatitis C virus agent. For example, an iRNA featured herein can be administered along with an interferon alpha for treatment of hepatitis C infection.

In one embodiment, the iRNA is COL1A1iRNA and it is administered in combination with a second iRNA targeting a second gene that is involved in signaling pathway leading to the excess deposition of collagen extracellularly in the liver. For example, the second gene can be the gene encoding TGFβ, SMAD2 or SMAD3.

In one embodiment, the iRNA is COL1A1iRNA, TGFβiRNA or SMAD2/3iRNA it is administered in combination with a second iRNA targeting a second gene that is involved in maturation of the pro-collagen produced by the activated HSCs, the collagen-specific chaperone, HSP47.

Patients can be administered a therapeutic amount of iRNA, such as 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The iRNA can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the iRNA can reduce COL1A1 levels, e.g., in a cell, tissue, e.g. liver tissue or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or IFN-alpha) levels.

Owing to the inhibitory effects on COL1A1 expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life and prolong survival in that irreversible liver damage can prevented.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples Example 1: Interference RNA (iRNA) Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Oligonucleotide Synthesis

Applicants have used several different methods to generate the iRNA molecules described herein. This Example describes one approach that has been used. The ordinarily skilled artisan can use any method known in the art to prepare iRNAs as described herein.

Oligonucleotides are synthesized on an AKTA oligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N₄-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N₂-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH₃CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10: 87: 3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection)

After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.

Analysis

The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotidess are diluted in water to 150 μL and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.

IRNA Preparation

For the general preparation of iRNA, equimolar amounts of sense and antisense strand are heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.

Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′- phosphodiester bonds. Abbreviation Nucleotide(s) A adenosine C cytidine G guanosine T thymidine U uridine N any nucleotide (G, A, C, T or U) a 2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u 2′-O-methyluridine dT 2′-deoxythymidine s phosphorothioate linkage

Example 2: COL1A1, TGFβ SMAD2/3 siRNAs Design and Synthesis Transcripts

Oligonucleotide designs were carried out to identify siRNAs targeting the genes encoding the human collagen1A1 molecule (COL1A1), the human transforming growth factor beta 1 (TGFβ), the SMAD family member 2 and 3, and the orthologous sequences from mice (Mus musculus) and rat (Rattus norvegicus). The design process used the human COL1A1 mRNA NM_000088.3 (SEQ ID NO: 172), the mouse COL1A1 mRNA NM_007742.3 (SEQ ID NO: 173), the human TGFβ mRNA NM_000660.4 (SEQ ID NO: 174), the mouse TGFβ mRNA NM_011577.1 (SEQ ID NO: 175), the four human SMAD3 mRNAs NM_001145102.1 (SEQ ID NO: 180), NM_001145103.1 (SEQ ID NO: 181), NM_001145104.1 (SEQ ID NO: 182), and NM_005902.3 (SEQ ID NO: 183), the mouse SMAD3 mRNAs NM_016769.4 (SEQ ID NO: 184), the three human SMAD2 mRNAs are NM_001003652.2 (SEQ ID NO: 176), NM_001135937.1 (SEQ ID NO: 177), and NM_005901.4 (SEQ ID NO: 178), and the mouse SMAD2 mRNAs is NM_010754.4. All sequences were obtained from the NCBI Refseq collection.

Two sets of oligos were designed: a human-specific set of oligos with 100% identity to human COL1A1, but less than 100% identity in mouse or rat, and a second set of siRNAs with 100% identity to the human, non-human primate, mouse and both rat COL1A1 transcripts. A set of oligos was designed with 100% identity to mouse SMAD2 (NM_010754) and mouse SMAD3 (NM_016769). For TGFβ, the oligo set was designed with 100% identity to human and mouse sequences. siRNA Design and Specificity Prediction

The specificity of the 19mer oligo sets was predicted from each sequence. The COL1A1, TGFβ AND SMAD2/3 siRNAs were used in a comprehensive search against their respective human, or mouse and rat transcriptomes (defined as NM_ and XM_ records within the NCBI Refseq set) using the FASTA algorithm. The Python script ‘offtargetFasta.py’ was then used to parse the alignments and generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. The off-target score is calculated as follows: mismatches between the oligo and the transcript are given penalties. A mismatch in the seed region in positions 2-9 of the oligo is given a penalty of 2.8; mismatches in the putative cleavage sites 10 and 11 are given a penalty of 1.2, and all other mismatches a penalty of 1. The off-target score for each oligo-transcript pair is then calculated by summing the mismatch penalties. The lowest off-target score from all the oligo-transcript pairs is then determined and used in subsequent sorting of oligos. Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderate specific. In picking which oligos to synthesize, we sorted from high to low by the off-target score of the antisense strand and took the best (lowest off-target score) oligo pairs.

Synthesis of COL1A1, TGFβ and SMAD2/3 RNA Sequences

COL1A1, TGFβ AND SMAD2/3 iRNA sequences were synthesized on a MerMade 192 synthesizer at 1 μmol scale.

For all the sequences in the list, ‘endolight’ chemistry was applied as detailed below.

-   -   All pyrimidines (cytosine and uridine) in the sense strand         contained 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)     -   In the antisense strand, pyrimidines adjacent to (towards 5′         position) ribo A nucleoside were replaced with their         corresponding 2-O-Methyl nucleosides     -   A two base dTsdT extension at 3′ end of both sense and anti         sense sequences was introduced     -   The sequence file was converted to a text file to make it         compatible for loading in the MerMade 192 synthesis software

Synthesis, Cleavage and Deprotection

The synthesis of COL1A1, TGFβ and SMAD2/3 RNA sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry.

The synthesis of the above sequences was performed at 1 um scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.

The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. The crude sequences were precipitated using acetone:ethanol (80:20) mix and the pellet were re-suspended in 0.02M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.

Purification and Desalting

COL1A1, TGFβ and SMAD2/3 RNA sequences were purified on AKTA explorer purification system using Source 15Q column. A column temperature of 65C was maintained during purification. Sample injection and collection was performed in 96 well (1.8 mL—deep well) plates. A single peak corresponding to the full length sequence was collected in the eluent. The purified sequences were desalted on a Sephadex G25 column using AKTA purifier. The desalted COL1A1, TGFβ and SMAD2/3 sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The single strands were then submitted for annealing.

Example 3: In Vitro Screening of COL1A1 siRNA in NIH3T3 Cells Cell Culture and Transfections

NIH3T3 cells were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in DMEM (INVITROGEN™) supplemented with 10% FBS before being released from the plate by trypsinization. Reverse transfection was carried out by adding 5 ul of Opti-MEM to 5 ul of siRNA duplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.20 of Lipofectamine RNAiMax per well (INVITROGEN™, Carlsbad Calif. cat #13778-150) and incubated at room temperature for 15 minutes. Cells were were added to the transfection mix and incubated for 24 hours prior to RNA purification. Experiments were performed at 10 nM final duplex concentration for single dose screens with each of the COL1A1 duplexes. A subset of 10 duplexes that showed robust silencing in the 10 nM single dose screens were assayed over a range of concentrations from 10 nM to 10 fM using serial dilutions to determine their IC₅₀. Selected AD 21349 (SEQ ID NO: 53 and SEQ ID NO: 54) has an IC₅₀ of 65 pM. The siRNA tested were cross-reactive for rodent, NHP, and human (See FIG. 1). Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Forer City CA, part #: AM1830)

Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads and Lysis/Binding Enhancer mixture were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 ul of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA was eluted with 50 μl of water. cDNA synthesis using ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.20 of H₂O per reaction were added into 10 μl total RNA. cDNA was generated using a MJ Research or Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR

20 of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl COL1A1, TGFβ or SMAD2/3 TaqMan probe and 5 μl Roche Probes Master Mix (Roche Cat #04887301001) in a total of 10 μl per well in a LightCycler 480 384 well plate (Roche cat #0472974001). Real time PCR was done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex was tested in at least two independent transfections. Each transfection was assayed by qPCR in duplicate.

Real time data were analyzed using the ΔΔCt method. Each sample was normalized to GAPDH expression and knockdown was assessed relative to cells transfected with the non-targeting duplex AD-1955. IC₅₀s were defined using a 4 parameter fit model in XLfit. As shown in FIG. 1, the efficacy of single doses of collagen1A1 siRNA in NIH-3T3 cells was examined in vitro. siRNA AD-21349 (SEQ ID NO: 53 and SEQ ID NO: 54), has an IC50 of ˜65 pM on the production of COL1A1 mRNA.

Example 4: Carbon Tetrachloride (CCl₄)-Induced Mouse Liver Fibrosis Models

In general, liver injury can be caused by many factors, for example, toxic chemicals such as carbon tetrachloride (CCl₄), diamethylnitrozamine and thioacetamide; damage due to immunological response in vivo, e. g. to heterologous serum and due to pathogens such as schistosomas and viruses; ligation of the common bile duct that leads to biliary fibrosis; and alcohol abuse. Examples of alcoholic liver injury animal models are the baboon placed on ethanol diet and the Tsukamoto/French model in rats.

The experimental mouse model use herein is one wherein liver injury is induced with the chemical CCl₄. This mouse model is a well-investigated chemical induced model of liver fibrosis. Hepatic pathological changes observed in CCl₄-treated mice include necrosis, inflammation, and fibrosis, along with increased serum alanine aminotransferase activity. Several microarray studies indicated CCl₄-induced up-regulation of genes involved in liver fibrogenesis, e.g. procollagen type 1A, which correlated well with prolong fibrotic changes in the liver (Jiang Y., et al., 2004, Toxicol. Sci., 79:404-410). Oral gavage of Balb/c animals with CCl₄ 1.75 ml/kg in mineral oil (MO), was administered on days 1 & 8. At 24 hours after the initial dose of CCl₄, the serum liver enzyme levels were measured. At 10 or 11 days, the animals were sacrificed and the histology of the livers was stained with Sirius red to determine the level of extracellular collagen.

The level of collagen expression was significantly increased, as were the two liver function biomarker enzymes, alanine transaminase (ALT) and aspartate transaminase (AST), within 24 of the initial dose of CCl₄.

Similarly, the CCl₄-treated mouse livers showed increased staining for extracellular collagen by way of Sirius red staining compared to control mineral oil treated animals.

Example 5: In Vivo Testing of COL1A1 siRNA in CCl₄ Mouse Model

To evaluate the AF09 and AF12-based delivery of siCOL1A1 for COL1A1 knock-down in the CCl₄ mouse model, liver injury in Balb/c animals was induced using CCl₄ by oral gavage (2×) on Day1 and Day 8 at a final dose of 1.75 ml/kg. Control animals were given mineral oil only. Subsequently, on Day 9, the mice were injected intravenously with the AF09 and AF12 formulated siRNAs and then killed at about 40 hours post siRNA injection on Day 11. Control siRNA used was siLUC (AD1955). The dosage of both siCOL1A1 (AD21349) (SEQ ID NO: 53 and SEQ ID NO: 54) and siLUC (AD1955) was 3 mg/kg. The COL1A1 and GAPDH mRNA levels in liver were analyzed using Taqman-QPCR. A total of 10-15 animals were used per group per condition. The COL1A1 mRNA level was normalized to that of the GAPDH mRNA level.

FIG. 2 illustrates the CCl₄/siRNA treatment regimen and FIG. 3 summarizes the results obtained. Both the AF09 and AF12 formulated siCOL1A1 (AD21349) (SEQ ID NO: 53 and SEQ ID NO: 54) significantly reduced the level of the COL1A1 mRNA level compared to the control siLUC siRNA in CCl₄-treated mice. In addition, COL1A1 knockdown with AF12-21349 (SEQ ID NO: 53 and SEQ ID NO: 54) was better than AF09-21349 at 3 mg/Kg.

To evaluate the dosage response of the AF12-based delivery siCOL1A1 in the CCl₄-treated mouse model, liver injury in Balb/c animals was induced using CCl₄ by oral gavage (2×) on Day 1 and Day 8 at a final dose of 1.75 ml/kg. Control animals were given mineral oil only. On Day 9, the mice were injected intravenously with the AF12 formulated siRNA at various dosages and then killed at about 40 hours post siRNA injection on Day 11. Control siRNA used was AF12_LUC (AD1955). The dosages of both AF12_COL1A1 (AD21349) (SEQ ID NO: 53 and SEQ ID NO: 54) and siLUC (AD1955) ranged from 0.1 mg/kg to 3 mg/kg. The COL1A1 and GAPDH mRNA levels in liver were analyzed using Taqman-QPCR. A total of 10 animals were used per group per dosage and 5 animals were used for the mineral oil control group. The COL1A1 mRNA level was normalized to that of the GAPDH mRNA level.

FIG. 4 illustrates the dosage response in the CCl₄/siRNA treatment regimen and FIG. 5 summarizes the results obtained. The AF12 COL1A1 (AD21349) (SEQ ID NO: 53 and SEQ ID NO: 54) formulation at dosages of 3 mg/kg, 1 mg/kg, and 0.5 mg/kg significantly reduced the level of the COL1A1 mRNA level as compared to the control siLUC siRNA in CCl₄ treated mice. The ED₅₀ was 0.1 mg/kg for COL1A1 knockdown with AF12-AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54). The data was normalized to AF12Luc.

To evaluate the AF11-based delivery of siCOL1A1 in the mouse model, liver injury in Balb/c animals was induced using CCl₄ by oral gavage (2×) on Day1 and Day 7 at a final dose of 1.75 ml/kg. Control animals were given mineral oil only. On Day 8, the mice were injected intravenously with the AF11 formulated siRNAs at various doses and then killed at about 40 hours post siRNA injection on Day 10. Control siRNA used was siLUC (AD1955). The dosage of both siCOL1A1 (AD21349) (SEQ ID NO: 53 and SEQ ID NO: 54) and siLUC (AD1955) was 3 mg/kg. The COL1A1 and GAPDH mRNA levels in liver were analyzed using Taqman-QPCR. A total of 10 animals were used per group per dosage, 5 animals for the mineral oil control group. The COL1A1 mRNA level was normalized to that of the GAPDH mRNA level.

FIG. 6 illustrates the CCl₄/AF11-SiRNA treatment regimen and FIG. 7 summarizes the results obtained. The AF11 COL1A1 (AD21349) (SEQ ID NO: 53 and SEQ ID NO: 54) formulation at dosage of 3 mg/kg significantly reduced the level of the COL1A1 mRNA level as compared to the control siLUC siRNA in CCl₄-treated mice. Approximately 65% COL1A1 mRNA knockdown was achieved with AF11-21349 formulation. The data was normalized to AF12 Luc.

Hepatic stellate cells (HSC) are major contributors to the abnormal matrix deposition in during liver fibrosis. These cells undergo transdifferentiation from a “quiescent” HSc into “activated” fibrogenic myofibroblasts both in chronic liver disease. The transdifferentiation is characterized by an increase in proliferation, changes in morphology with expression of α-smooth muscle actin (α-SMA), and the deposition of extracellular matrix proteins, fibrillar collgen (Friedman et al., 1992, Hepatology, 15:234-243; Friedman, 2000, J. Biol. Chem., 275:2247-2250). Uemura M. et al., (2005, Mol. Biol. Cell, 16:4214-4224) demonstrated that SMAD3 overexpression in primary rat HSC and treating primary rat HSC with TGFβ led to increased focal adhesion and α-SMA organization. This indicates that TGFβ, signalling via SMAD3, plays an important role in the morphological and functional maturation of the “activated” HSC or myofibroblasts.

To confirm that the AF-based (i. e. AF09, AF11, AF12-based formulation) delivery method did deliver the siRNA to the “activated” HSC or myofibroblasts in the CCl₄-treated mice, the knock-down of α-SMA, which is also overexpressed in the “activated” HSC, was determined using AF09_ACTA2. α-SMA was selected as a target because it is present in activated myofibroblasts, and vascular smooth muscle cells but is absent in hepatocytes. FIG. 8 showed ACTA2 expression knock-down in the liver cells of the CCl₄-treated mice. This data confirmed the delivery of AF09-ACTA2 to HSC in the CCl₄-treated mice.

Example 6: In Vitro Screening of TGFβ siRNA in NIH3T3 Cells

NIH3T3 cells were grown, cultured and treated with siRNA as described in Example 3 except the siRNAs applied were targeted to the transforming growth factor beta 1 (TGFβ) gene. Experiments were performed at 10 nM final duplex concentration for single dose screens with each of the TGFβ duplexes (FIG. 9).

A subset of 10 duplexes that showed robust silencing in the 10 nM single dose screens was assayed over a range of concentrations from 10 nM to 10 fM using serial dilutions to determine their IC₅₀. Selected siRNA AD22149 (SEQ ID NO: 91 and SEQ ID NO: 92) and AD22138 (SEQ ID NO: 69 and SEQ ID NO: 70) have an IC₅₀ of ˜20 pM and ˜70 pM respectively on the mRNA of TGFβ. The siRNA tested were cross-reactive for rodent, NHP, and human.

Example 7: In Vitro Screening of SMAD2 siRNA in NIH3T3 Cells

NIH3T3 cells were grown, cultured and treated with siRNA as described in Example 3 except the siRNAs applied were targeted to the SMAD family member 2 and 3 (SMAD2 and SMAD3) genes. Experiments were performed at 10 nM final duplex concentration for single dose screens with each of the SMAD2/3 duplexes (FIG. 10).

A subset of 10 duplexes that showed robust silencing in the 10 nM single dose screens was assayed over a range of concentrations from 10 nM to 10 fM using serial dilutions to determine their IC₅₀. Selected siRNA AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141) has an IC₅₀ of ˜75 pM on the mRNA of SMAD2.

Example 8: In Vivo Testing of SMAD2 siRNA in CCl₄ Mouse Model

Because SMADs are involved in the TGFβ signaling in fibrosis development, it was examined whether if knocking down SMAD expression would affect COL1A1 expression in the liver injury mouse model. To evaluate the LNP-based delivery of siSMAD2 AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141) for SMAD2 knock-down in the CCl₄ mouse model, liver injury in Balb/c animals was induced using CCl₄ by oral gavage (2×) on Day 0 at a dose of 1.75 ml/kg. Control animals were given mineral oil only. On Day 2, the mice were injected intravenously with the LNP-formulated siRNAs and then killed on Day 4 post CCl₄ induction. Control siRNA was siLUC (AD1955). The dosage of both siSMAD2/3 AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141) and siLUC (AD1955) was 3 mg/kg. The SMAD2 and GAPDH mRNA levels in liver were analyzed using Taqman-QPCR. A total of 10 animals were used per group per condition. The SMAD2 mRNA level was normalized to that of the GAPDH mRNA level.

FIG. 11 summarizes the results obtained. The siSMAD2/3 AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141) significantly reduced the level of the SMAD2/3 mRNA level as compared to the control siLUC siRNA in the CCl₄-treated mice. The reduction in SMAD2 expression was approximately 80%.

To evaluate the LNP-based delivery of siSMAD2 AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141) for SMAD2 knock-down and its effect on COL1A1 expression in the CCl₄ mouse model, liver injury in Balb/c animals was induced using CCl₄ by oral gavage (2×) on Day 0 at a dose of 1.75 ml/kg. Control animals were given mineral oil only. On Day 2, the mice were injected intravenously with the LNP-formulated siRNAs and then killed on Day 4 post CCl₄ induction. Control siRNA was siLUC (AD1955). The dosage of both siSMAD2/3 AD-20916 (SEQ ID NO: 140 and SEQ ID NO: 141) and siLUC (AD1955) was 3 mg/Kg. The SMAD2, COL1A1 and GAPDH mRNA levels in liver were analyzed using Taqman-QPCR. A total of 10 animals were used per group per condition. The SMAD2 mRNA level was normalized to that of the GAPDH mRNA level.

FIG. 12 summarizes the results obtained. SMAD2 knock-down using the LNP-based delivery of siSMAD2 AD-20916 led to a decrease in the expression of COL1A1 in the CCl₄-treated mice. The decrease was about 50-60% relative to the Luc siRNA injected animals.

Example 9: In Vivo Demonstration of COL1A1 siRNA Inhibition in a Mouse Model of Chronic Liver Disease

Chronic liver disease is caused by repeated insults to the liver, resulting in a progressive process of liver destruction and regeneration, that ultimately leads to liver fibrosis and cirrhosis. To evaluate whether effective inhibition of COL1A1 can be mediated under such repeated insults to the liver, and whether such treatment is effective in reducing fibrosis under such circumstances, a mouse model of chronic liver injury was established involving multiple-insults, and the effects of repeated administration of siRNA against COL1A1 for COL1A1 knock-down were determined. In this mouse model of chronic liver injury, liver injury in Balb/c animals was induced using CCl₄ by oral gavage on Day 1, Day 8, Day 15, Day 22, Day 29, and Day 36 at a final dose of 1.75 ml/kg. Control animals were given mineral oil only on these same days. On Day 23, Day 27, Day 30, Day 33, and Day 37 the mice were injected intravenously with the AF09 and AF12 formulated COL1A1 siRNAs and then killed at about 40-48 hours post the final siRNA injection, i.e., on Day 39. Control siRNA used was siLUC (AD1955). The dosage of both siCOL1A1 (AD21349) (SEQ ID NO: 53 and SEQ ID NO: 54) and siLUC (AD1955) was 1 mg/kg. The COL1A1 and GAPDH mRNA levels in liver were analyzed using Taqman-QPCR. A total of 10 animals were used per group per condition. The COL1A1 mRNA level was normalized to that of the GAPDH mRNA level.

FIG. 13 summarizes the results obtained. Surprisingly, COL1A1 knock-down using the C12-based delivery of siCOL1A1 (AD21349) (SEQ ID NO: 53 and SEQ ID NO: 54) led to a significant and unexpected decrease in the expression of COL1A1 in mice receiving multiple (×6) liver insults over a 39 day period. The decrease was about 95% relative to the Luc siRNA injected animals.

Example 10: In Vivo Demonstration of COL1A1 siRNA Inhibition in Mouse Models of Chronic Liver Disease (CCl₄ Model, TAA Model, and Bile Duct Ligation Model)

The CCl₄ model leads to liver damage that is severely shifted to the pericentral region only, and thus is a good model of liver damage to the pericentral region of the liver.

The TAA model leads to uniform liver damage (i.e., pericentral and periportal). The TAA model is slower than CCl₄, and thus is considered to more closely mimic liver injury developing in the human population.

The bile duct-ligated model is mediated by ligation or cannulation of bile-duct. This model effects the periductal regions and is a good model for biliary fibrosis in humans. In contrast to the other two models, the bile duct ligation model results in continued and progressive injury, i.e., chronic liver disease.

FIG. 16 demonstrates experimental design and efficacy of COL1A1 knock-down in a bile duct ligation mouse model using AF12-based delivery of siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

FIG. 17 shows relative Col1a1 expression in bile duct ligated animals.

FIG. 18 demonstrates efficacy of COL1A1 knock-down in a bile duct ligation mouse model using siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

FIG. 19 demonstrates experimental design and efficacy of COL1A1 knock-down in a TAA mouse model of liver injury using siCOL1A1 AD21349 (SEQ ID NO: 53 and SEQ ID NO: 54).

TABLE 2 Exemplary diseases in which fibrosis can be reduced by treating the underlying disorder Disease Therapy Hepatitis B Lamivudine, others Hepatitis C Interferon alpha Autoimmune hepatitis Corticosteroids Bile duct obstruction Surgical decompression Hemochromatosis Iron depletion Alcoholic hepatitis ^(b) Corticosteroids Primary binary cirrhosis ^(b) Ursodeoxycholic acid, MTX Non-alcholic PPAR gamma ligands steatohepatitisc ^(c)

TABLE 3 COL1A1 siRNA Duplexes Sense and Corresponding Antisense Duplexes Sense and Corresponding Antisense Duplexes Corresponding Corresponding Sense Strand Antisense Strand Sense Strand Antisense Strand SEQ ID NO: 1 sense SEQ ID NO: 2 antisense SEQ ID NO: 3 sense SEQ ID NO: 4 antisense SEQ ID NO: 5 sense SEQ ID NO: 6 antisense SEQ ID NO: 7 sense SEQ ID NO: 8 antisense SEQ ID NO: 9 sense SEQ ID NO: 10 antisense SEQ ID NO: 11 sense SEQ ID NO: 12 antisense SEQ ID NO: 13 sense SEQ ID NO: 14 antisense SEQ ID NO: 15 sense SEQ ID NO: 16 antisense SEQ ID NO: 17 sense SEQ ID NO: 18 antisense SEQ ID NO: 19 sense SEQ ID NO: 20 antisense SEQ ID NO: 21 sense SEQ ID NO: 22 antisense SEQ ID NO: 23 sense SEQ ID NO: 24 antisense SEQ ID NO: 25 sense SEQ ID NO: 26 antisense SEQ ID NO: 27 sense SEQ ID NO: 28 antisense SEQ ID NO: 29 sense SEQ ID NO: 30 antisense SEQ ID NO: 31 sense SEQ ID NO: 32 antisense SEQ ID NO: 33 sense SEQ ID NO: 34 antisense SEQ ID NO: 35 sense SEQ ID NO: 36 antisense SEQ ID NO: 37 sense SEQ ID NO: 38 antisense SEQ ID NO: 39 sense SEQ ID NO: 40 antisense SEQ ID NO: 41 sense SEQ ID NO: 42 antisense SEQ ID NO: 43 sense SEQ ID NO: 44 antisense SEQ ID NO: 45 sense SEQ ID NO: 46 antisense SEQ ID NO: 47 sense SEQ ID NO: 48 antisense SEQ ID NO: 49 sense SEQ ID NO: 50 antisense SEQ ID NO: 51 sense SEQ ID NO: 52 antisense SEQ ID NO: 53 sense SEQ ID NO: 54 antisense SEQ ID NO: 55 sense SEQ ID NO: 56 antisense SEQ ID NO: 57 sense SEQ ID NO: 58 antisense SEQ ID NO: 59 sense SEQ ID NO: 60 antisense SEQ ID NO: 61 sense SEQ ID NO: 62 antisense

TABLE 4 TGF-β siRNA duplexes Sense and Corresponding Antisense Duplexes Sense and Corresponding Antisense Duplexes Corresponding Corresponding Sense Strand Antisense Strand Sense Strand Antisense Strand SEQ ID NO: 63 sense SEQ ID NO: 64 antisense SEQ ID NO: 65 sense SEQ ID NO: 66 antisense SEQ ID NO: 67 sense SEQ ID NO: 68 antisense SEQ ID NO: 69 sense SEQ ID NO: 70 antisense SEQ ID NO: 71 sense SEQ ID NO: 72 antisense SEQ ID NO: 73 sense SEQ ID NO: 74 antisense SEQ ID NO: 75 sense SEQ ID NO: 76 antisense SEQ ID NO: 77 sense SEQ ID NO: 78 antisense SEQ ID NO: 79 sense SEQ ID NO: 80 antisense SEQ ID NO: 81 sense SEQ ID NO: 82 antisense SEQ ID NO: 83 sense SEQ ID NO: 84 antisense SEQ ID NO: 85 sense SEQ ID NO: 86 antisense SEQ ID NO: 87 sense SEQ ID NO: 88 antisense SEQ ID NO: 89 sense SEQ ID NO: 90 antisense SEQ ID NO: 91 sense SEQ ID NO: 92 antisense SEQ ID NO: 93 sense SEQ ID NO: 94 antisense SEQ ID NO: 95 sense SEQ ID NO: 96 antisense SEQ ID NO: 97 sense SEQ ID NO: 98 antisense SEQ ID NO: 99 sense SEQ ID NO: 100 antisense SEQ ID NO: 101 sense SEQ ID NO: 102 antisense SEQ ID NO: 103 sense SEQ ID NO: 104 antisense SEQ ID NO: 105 sense SEQ ID NO: 106 antisense SEQ ID NO: 107 sense SEQ ID NO: 108 antisense SEQ ID NO: 109 sense SEQ ID NO: 110 antisense SEQ ID NO: 111 sense SEQ ID NO: 112 antisense SEQ ID NO: 113 sense SEQ ID NO: 114 antisense SEQ ID NO: 115 sense SEQ ID NO: 116 antisense SEQ ID NO: 117 sense SEQ ID NO: 118 antisense SEQ ID NO: 119 sense SEQ ID NO: 120 antisense SEQ ID NO: 121 sense SEQ ID NO: 122 antisense SEQ ID NO: 123 sense SEQ ID NO: 124 antisense SEQ ID NO: 125 sense SEQ ID NO: 126 antisense SEQ ID NO: 127 sense SEQ ID NO: 128 antisense SEQ ID NO: 129 sense SEQ ID NO: 130 antisense

TABLE 5 SMAD2/3 siRNA duplexes Sense and Corresponding Antisense Duplexes Sense and Corresponding Antisense Duplexes Corresponding Corresponding Sense Strand Antisense Strand Sense Strand Antisense Strand SEQ ID NO: 132 sense SEQ ID NO: 133 antisense SEQ ID NO: 134 sense SEQ ID NO: 135 antisense SEQ ID NO: 136 sense SEQ ID NO: 137 antisense SEQ ID NO: 138 sense SEQ ID NO: 139 antisense SEQ ID NO: 140 sense SEQ ID NO: 141 antisense SEQ ID NO: 142 sense SEQ ID NO: 143 antisense SEQ ID NO: 144 sense SEQ ID NO: 145 antisense SEQ ID NO: 146 sense SEQ ID NO: 147 antisense SEQ ID NO: 148 sense SEQ ID NO: 149 antisense SEQ ID NO: 150 sense SEQ ID NO: 151 antisense SEQ ID NO: 152 sense SEQ ID NO: 153 antisense SEQ ID NO: 154 sense SEQ ID NO: 155 antisense SEQ ID NO: 156 sense SEQ ID NO: 157 antisense SEQ ID NO: 158 sense SEQ ID NO: 159 antisense SEQ ID NO: 160 sense SEQ ID NO: 161 antisense SEQ ID NO: 162 sense SEQ ID NO: 163 antisense SEQ ID NO: 164 sense SEQ ID NO: 165 antisense SEQ ID NO: 166 sense SEQ ID NO: 167 antisense

TABLE 6 Human COL1A1 iRNA Sequences Designed on the Basis of the Human COL1A1 Transcript NM_000088.3 (SEQ ID NO: 172) that are Cross-Reactive with Mouse Col1a1 Transcript NM_007742.3 (SEQ ID NO: 173) Sense and Corresponding Antisense Duplexes Sense and Corresponding Antisense Duplexes Corresponding Corresponding Sense Strand Antisense Strand Sense Strand Antisense Strand SEQ ID NO: 185 sense SEQ ID NO: 186 antisense SEQ ID NO: 187 sense SEQ ID NO: 188 antisense SEQ ID NO: 189 sense SEQ ID NO: 190 antisense SEQ ID NO: 191 sense SEQ ID NO: 192 antisense SEQ ID NO: 193 sense SEQ ID NO: 194 antisense SEQ ID NO: 195 sense SEQ ID NO: 196 antisense SEQ ID NO: 197 sense SEQ ID NO: 198 antisense SEQ ID NO: 199 sense SEQ ID NO: 200 antisense SEQ ID NO: 201 sense SEQ ID NO: 202 antisense SEQ ID NO: 203 sense SEQ ID NO: 204 antisense SEQ ID NO: 205 sense SEQ ID NO: 206 antisense SEQ ID NO: 207 sense SEQ ID NO: 208 antisense SEQ ID NO: 209 sense SEQ ID NO: 210 antisense SEQ ID NO: 211 sense SEQ ID NO: 212 antisense SEQ ID NO: 213 sense SEQ ID NO: 214 antisense SEQ ID NO: 215 sense SEQ ID NO: 216 antisense SEQ ID NO: 217 sense SEQ ID NO: 218 antisense SEQ ID NO: 219 sense SEQ ID NO: 220 antisense SEQ ID NO: 221 sense SEQ ID NO: 222 antisense SEQ ID NO: 223 sense SEQ ID NO: 224 antisense SEQ ID NO: 225 sense SEQ ID NO: 226 antisense SEQ ID NO: 227 sense SEQ ID NO: 228 antisense SEQ ID NO: 229 sense SEQ ID NO: 230 antisense SEQ ID NO: 231 sense SEQ ID NO: 232 antisense SEQ ID NO: 233 sense SEQ ID NO: 234 antisense SEQ ID NO: 235 sense SEQ ID NO: 236 antisense SEQ ID NO: 237 sense SEQ ID NO: 238 antisense SEQ ID NO: 239 sense SEQ ID NO: 240 antisense SEQ ID NO: 241 sense SEQ ID NO: 242 antisense SEQ ID NO: 243 sense SEQ ID NO: 244 antisense SEQ ID NO: 245 sense SEQ ID NO: 246 antisense SEQ ID NO: 247 sense SEQ ID NO: 248 antisense SEQ ID NO: 249 sense SEQ ID NO: 250 antisense SEQ ID NO: 251 sense SEQ ID NO: 252 antisense SEQ ID NO: 253 sense SEQ ID NO: 254 antisense SEQ ID NO: 255 sense SEQ ID NO: 256 antisense SEQ ID NO: 257 sense SEQ ID NO: 258 antisense SEQ ID NO: 259 sense SEQ ID NO: 260 antisense SEQ ID NO: 261 sense SEQ ID NO: 262 antisense SEQ ID NO: 263 sense SEQ ID NO: 264 antisense SEQ ID NO: 265 sense SEQ ID NO: 266 antisense SEQ ID NO: 267 sense SEQ ID NO: 268 antisense SEQ ID NO: 269 sense SEQ ID NO: 270 antisense SEQ ID NO: 271 sense SEQ ID NO: 272 antisense SEQ ID NO: 273 sense SEQ ID NO: 274 antisense SEQ ID NO: 275 sense SEQ ID NO: 276 antisense SEQ ID NO: 277 sense SEQ ID NO: 278 antisense SEQ ID NO: 279 sense SEQ ID NO: 280 antisense SEQ ID NO: 281 sense SEQ ID NO: 282 antisense SEQ ID NO: 283 sense SEQ ID NO: 284 antisense SEQ ID NO: 285 sense SEQ ID NO: 286 antisense SEQ ID NO: 287 sense SEQ ID NO: 288 antisense SEQ ID NO: 289 sense SEQ ID NO: 290 antisense SEQ ID NO: 291 sense SEQ ID NO: 292 antisense SEQ ID NO: 293 sense SEQ ID NO: 294 antisense SEQ ID NO: 295 sense SEQ ID NO: 296 antisense SEQ ID NO: 297 sense SEQ ID NO: 298 antisense SEQ ID NO: 299 sense SEQ ID NO: 300 antisense SEQ ID NO: 301 sense SEQ ID NO: 302 antisense SEQ ID NO: 303 sense SEQ ID NO: 304 antisense SEQ ID NO: 305 sense SEQ ID NO: 306 antisense SEQ ID NO: 307 sense SEQ ID NO: 308 antisense SEQ ID NO: 309 sense SEQ ID NO: 310 antisense SEQ ID NO: 311 sense SEQ ID NO: 312 antisense SEQ ID NO: 313 sense SEQ ID NO: 314 antisense SEQ ID NO: 315 sense SEQ ID NO: 316 antisense SEQ ID NO: 317 sense SEQ ID NO: 318 antisense SEQ ID NO: 319 sense SEQ ID NO: 320 antisense SEQ ID NO: 321 sense SEQ ID NO: 322 antisense SEQ ID NO: 323 sense SEQ ID NO: 324 antisense SEQ ID NO: 325 sense SEQ ID NO: 326 antisense SEQ ID NO: 327 sense SEQ ID NO: 328 antisense SEQ ID NO: 329 sense SEQ ID NO: 330 antisense SEQ ID NO: 331 sense SEQ ID NO: 332 antisense SEQ ID NO: 333 sense SEQ ID NO: 334 antisense SEQ ID NO: 335 sense SEQ ID NO: 336 antisense SEQ ID NO: 337 sense SEQ ID NO: 338 antisense SEQ ID NO: 339 sense SEQ ID NO: 340 antisense SEQ ID NO: 341 sense SEQ ID NO: 342 antisense SEQ ID NO: 343 sense SEQ ID NO: 344 antisense SEQ ID NO: 345 sense SEQ ID NO: 346 antisense SEQ ID NO: 347 sense SEQ ID NO: 348 antisense SEQ ID NO: 349 sense SEQ ID NO: 350 antisense SEQ ID NO: 351 sense SEQ ID NO: 352 antisense SEQ ID NO: 353 sense SEQ ID NO: 354 antisense SEQ ID NO: 355 sense SEQ ID NO: 356 antisense SEQ ID NO: 357 sense SEQ ID NO: 358 antisense SEQ ID NO: 359 sense SEQ ID NO: 360 antisense SEQ ID NO: 361 sense SEQ ID NO: 362 antisense SEQ ID NO: 363 sense SEQ ID NO: 364 antisense SEQ ID NO: 365 sense SEQ ID NO: 366 antisense SEQ ID NO: 367 sense SEQ ID NO: 368 antisense SEQ ID NO: 369 sense SEQ ID NO: 370 antisense SEQ ID NO: 371 sense SEQ ID NO: 372 antisense SEQ ID NO: 373 sense SEQ ID NO: 374 antisense SEQ ID NO: 375 sense SEQ ID NO: 376 antisense SEQ ID NO: 377 sense SEQ ID NO: 378 antisense SEQ ID NO: 379 sense SEQ ID NO: 380 antisense SEQ ID NO: 381 sense SEQ ID NO: 382 antisense SEQ ID NO: 383 sense SEQ ID NO: 384 antisense SEQ ID NO: 385 sense SEQ ID NO: 386 antisense SEQ ID NO: 387 sense SEQ ID NO: 388 antisense SEQ ID NO: 389 sense SEQ ID NO: 390 antisense SEQ ID NO: 391 sense SEQ ID NO: 392 antisense SEQ ID NO: 393 sense SEQ ID NO: 394 antisense SEQ ID NO: 395 sense SEQ ID NO: 396 antisense SEQ ID NO: 397 sense SEQ ID NO: 398 antisense SEQ ID NO: 399 sense SEQ ID NO: 400 antisense SEQ ID NO: 401 sense SEQ ID NO: 402 antisense SEQ ID NO: 403 sense SEQ ID NO: 404 antisense SEQ ID NO: 405 sense SEQ ID NO: 406 antisense SEQ ID NO: 407 sense SEQ ID NO: 408 antisense SEQ ID NO: 409 sense SEQ ID NO: 410 antisense SEQ ID NO: 411 sense SEQ ID NO: 412 antisense SEQ ID NO: 413 sense SEQ ID NO: 414 antisense SEQ ID NO: 415 sense SEQ ID NO: 416 antisense SEQ ID NO: 417 sense SEQ ID NO: 418 antisense SEQ ID NO: 419 sense SEQ ID NO: 420 antisense SEQ ID NO: 421 sense SEQ ID NO: 422 antisense SEQ ID NO: 423 sense SEQ ID NO: 424 antisense SEQ ID NO: 425 sense SEQ ID NO: 426 antisense SEQ ID NO: 427 sense SEQ ID NO: 428 antisense SEQ ID NO: 429 sense SEQ ID NO: 430 antisense SEQ ID NO: 431 sense SEQ ID NO: 432 antisense SEQ ID NO: 433 sense SEQ ID NO: 434 antisense SEQ ID NO: 435 sense SEQ ID NO: 436 antisense SEQ ID NO: 437 sense SEQ ID NO: 438 antisense SEQ ID NO: 439 sense SEQ ID NO: 440 antisense SEQ ID NO: 441 sense SEQ ID NO: 442 antisense SEQ ID NO: 443 sense SEQ ID NO: 444 antisense SEQ ID NO: 445 sense SEQ ID NO: 446 antisense SEQ ID NO: 447 sense SEQ ID NO: 448 antisense SEQ ID NO: 449 sense SEQ ID NO: 450 antisense SEQ ID NO: 451 sense SEQ ID NO: 452 antisense SEQ ID NO: 453 sense SEQ ID NO: 454 antisense SEQ ID NO: 455 sense SEQ ID NO: 456 antisense SEQ ID NO: 457 sense SEQ ID NO: 458 antisense SEQ ID NO: 459 sense SEQ ID NO: 460 antisense SEQ ID NO: 461 sense SEQ ID NO: 462 antisense SEQ ID NO: 463 sense SEQ ID NO: 464 antisense SEQ ID NO: 465 sense SEQ ID NO: 466 antisense SEQ ID NO: 467 sense SEQ ID NO: 468 antisense SEQ ID NO: 469 sense SEQ ID NO: 470 antisense SEQ ID NO: 471 sense SEQ ID NO: 472 antisense SEQ ID NO: 473 sense SEQ ID NO: 474 antisense SEQ ID NO: 475 sense SEQ ID NO: 476 antisense SEQ ID NO: 477 sense SEQ ID NO: 478 antisense SEQ ID NO: 479 sense SEQ ID NO: 480 antisense SEQ ID NO: 481 sense SEQ ID NO: 482 antisense SEQ ID NO: 483 sense SEQ ID NO: 484 antisense SEQ ID NO: 485 sense SEQ ID NO: 486 antisense SEQ ID NO: 487 sense SEQ ID NO: 488 antisense SEQ ID NO: 489 sense SEQ ID NO: 490 antisense SEQ ID NO: 491 sense SEQ ID NO: 492 antisense SEQ ID NO: 493 sense SEQ ID NO: 494 antisense SEQ ID NO: 495 sense SEQ ID NO: 496 antisense SEQ ID NO: 497 sense SEQ ID NO: 498 antisense SEQ ID NO: 499 sense SEQ ID NO: 500 antisense SEQ ID NO: 501 sense SEQ ID NO: 502 antisense SEQ ID NO: 503 sense SEQ ID NO: 504 antisense SEQ ID NO: 505 sense SEQ ID NO: 506 antisense SEQ ID NO: 507 sense SEQ ID NO: 508 antisense SEQ ID NO: 509 sense SEQ ID NO: 510 antisense SEQ ID NO: 511 sense SEQ ID NO: 512 antisense SEQ ID NO: 513 sense SEQ ID NO: 514 antisense SEQ ID NO: 515 sense SEQ ID NO: 516 antisense SEQ ID NO: 517 sense SEQ ID NO: 518 antisense SEQ ID NO: 519 sense SEQ ID NO: 520 antisense SEQ ID NO: 521 sense SEQ ID NO: 522 antisense SEQ ID NO: 523 sense SEQ ID NO: 524 antisense SEQ ID NO: 525 sense SEQ ID NO: 526 antisense SEQ ID NO: 527 sense SEQ ID NO: 528 antisense SEQ ID NO: 529 sense SEQ ID NO: 530 antisense SEQ ID NO: 531 sense SEQ ID NO: 532 antisense SEQ ID NO: 533 sense SEQ ID NO: 534 antisense SEQ ID NO: 535 sense SEQ ID NO: 536 antisense SEQ ID NO: 537 sense SEQ ID NO: 538 antisense SEQ ID NO: 539 sense SEQ ID NO: 540 antisense SEQ ID NO: 541 sense SEQ ID NO: 542 antisense SEQ ID NO: 543 sense SEQ ID NO: 544 antisense SEQ ID NO: 545 sense SEQ ID NO: 546 antisense SEQ ID NO: 547 sense SEQ ID NO: 548 antisense SEQ ID NO: 549 sense SEQ ID NO: 550 antisense SEQ ID NO: 551 sense SEQ ID NO: 552 antisense SEQ ID NO: 553 sense SEQ ID NO: 554 antisense SEQ ID NO: 555 sense SEQ ID NO: 556 antisense SEQ ID NO: 557 sense SEQ ID NO: 558 antisense SEQ ID NO: 559 sense SEQ ID NO: 560 antisense SEQ ID NO: 561 sense SEQ ID NO: 562 antisense SEQ ID NO: 563 sense SEQ ID NO: 564 antisense SEQ ID NO: 565 sense SEQ ID NO: 566 antisense SEQ ID NO: 567 sense SEQ ID NO: 568 antisense SEQ ID NO: 569 sense SEQ ID NO: 570 antisense SEQ ID NO: 571 sense SEQ ID NO: 572 antisense SEQ ID NO: 573 sense SEQ ID NO: 574 antisense SEQ ID NO: 575 sense SEQ ID NO: 576 antisense SEQ ID NO: 577 sense SEQ ID NO: 578 antisense SEQ ID NO: 579 sense SEQ ID NO: 580 antisense SEQ ID NO: 581 sense SEQ ID NO: 582 antisense SEQ ID NO: 583 sense SEQ ID NO: 584 antisense SEQ ID NO: 585 sense SEQ ID NO: 586 antisense SEQ ID NO: 587 sense SEQ ID NO: 588 antisense SEQ ID NO: 589 sense SEQ ID NO: 590 antisense SEQ ID NO: 591 sense SEQ ID NO: 592 antisense SEQ ID NO: 593 sense SEQ ID NO: 594 antisense SEQ ID NO: 595 sense SEQ ID NO: 596 antisense SEQ ID NO: 597 sense SEQ ID NO: 598 antisense SEQ ID NO: 599 sense SEQ ID NO: 600 antisense SEQ ID NO: 601 sense SEQ ID NO: 602 antisense SEQ ID NO: 603 sense SEQ ID NO: 604 antisense SEQ ID NO: 605 sense SEQ ID NO: 606 antisense SEQ ID NO: 607 sense SEQ ID NO: 608 antisense SEQ ID NO: 609 sense SEQ ID NO: 610 antisense SEQ ID NO: 611 sense SEQ ID NO: 612 antisense SEQ ID NO: 613 sense SEQ ID NO: 614 antisense SEQ ID NO: 615 sense SEQ ID NO: 616 antisense SEQ ID NO: 617 sense SEQ ID NO: 618 antisense SEQ ID NO: 619 sense SEQ ID NO: 620 antisense SEQ ID NO: 621 sense SEQ ID NO: 622 antisense SEQ ID NO: 623 sense SEQ ID NO: 624 antisense SEQ ID NO: 625 sense SEQ ID NO: 626 antisense SEQ ID NO: 627 sense SEQ ID NO: 628 antisense SEQ ID NO: 629 sense SEQ ID NO: 630 antisense SEQ ID NO: 631 sense SEQ ID NO: 632 antisense SEQ ID NO: 633 sense SEQ ID NO: 634 antisense SEQ ID NO: 635 sense SEQ ID NO: 636 antisense SEQ ID NO: 637 sense SEQ ID NO: 638 antisense SEQ ID NO: 639 sense SEQ ID NO: 640 antisense SEQ ID NO: 641 sense SEQ ID NO: 642 antisense SEQ ID NO: 643 sense SEQ ID NO: 644 antisense SEQ ID NO: 645 sense SEQ ID NO: 646 antisense SEQ ID NO: 647 sense SEQ ID NO: 648 antisense SEQ ID NO: 649 sense SEQ ID NO: 650 antisense SEQ ID NO: 651 sense SEQ ID NO: 652 antisense SEQ ID NO: 653 sense SEQ ID NO: 654 antisense SEQ ID NO: 655 sense SEQ ID NO: 656 antisense SEQ ID NO: 657 sense SEQ ID NO: 658 antisense SEQ ID NO: 659 sense SEQ ID NO: 660 antisense SEQ ID NO: 661 sense SEQ ID NO: 662 antisense SEQ ID NO: 663 sense SEQ ID NO: 664 antisense SEQ ID NO: 665 sense SEQ ID NO: 666 antisense SEQ ID NO: 667 sense SEQ ID NO: 668 antisense SEQ ID NO: 669 sense SEQ ID NO: 670 antisense SEQ ID NO: 671 sense SEQ ID NO: 672 antisense SEQ ID NO: 673 sense SEQ ID NO: 674 antisense SEQ ID NO: 675 sense SEQ ID NO: 676 antisense SEQ ID NO: 677 sense SEQ ID NO: 678 antisense SEQ ID NO: 679 sense SEQ ID NO: 680 antisense SEQ ID NO: 681 sense SEQ ID NO: 682 antisense SEQ ID NO: 683 sense SEQ ID NO: 684 antisense SEQ ID NO: 685 sense SEQ ID NO: 686 antisense SEQ ID NO: 687 sense SEQ ID NO: 688 antisense SEQ ID NO: 689 sense SEQ ID NO: 690 antisense SEQ ID NO: 691 sense SEQ ID NO: 692 antisense SEQ ID NO: 693 sense SEQ ID NO: 694 antisense SEQ ID NO: 695 sense SEQ ID NO: 696 antisense SEQ ID NO: 697 sense SEQ ID NO: 698 antisense SEQ ID NO: 699 sense SEQ ID NO: 700 antisense SEQ ID NO: 701 sense SEQ ID NO: 702 antisense SEQ ID NO: 703 sense SEQ ID NO: 704 antisense SEQ ID NO: 705 sense SEQ ID NO: 706 antisense SEQ ID NO: 707 sense SEQ ID NO: 708 antisense SEQ ID NO: 709 sense SEQ ID NO: 710 antisense SEQ ID NO: 711 sense SEQ ID NO: 712 antisense SEQ ID NO: 713 sense SEQ ID NO: 714 antisense SEQ ID NO: 715 sense SEQ ID NO: 716 antisense SEQ ID NO: 717 sense SEQ ID NO: 718 antisense SEQ ID NO: 719 sense SEQ ID NO: 720 antisense SEQ ID NO: 721 sense SEQ ID NO: 722 antisense SEQ ID NO: 723 sense SEQ ID NO: 724 antisense SEQ ID NO: 725 sense SEQ ID NO: 726 antisense SEQ ID NO: 727 sense SEQ ID NO: 728 antisense SEQ ID NO: 729 sense SEQ ID NO: 730 antisense SEQ ID NO: 731 sense SEQ ID NO: 732 antisense SEQ ID NO: 733 sense SEQ ID NO: 734 antisense SEQ ID NO: 735 sense SEQ ID NO: 736 antisense SEQ ID NO: 737 sense SEQ ID NO: 738 antisense SEQ ID NO: 739 sense SEQ ID NO: 740 antisense SEQ ID NO: 741 sense SEQ ID NO: 742 antisense SEQ ID NO: 743 sense SEQ ID NO: 744 antisense SEQ ID NO: 745 sense SEQ ID NO: 746 antisense SEQ ID NO: 747 sense SEQ ID NO: 748 antisense SEQ ID NO: 749 sense SEQ ID NO: 750 antisense SEQ ID NO: 751 sense SEQ ID NO: 752 antisense SEQ ID NO: 753 sense SEQ ID NO: 754 antisense SEQ ID NO: 755 sense SEQ ID NO: 756 antisense SEQ ID NO: 757 sense SEQ ID NO: 758 antisense SEQ ID NO: 759 sense SEQ ID NO: 760 antisense SEQ ID NO: 761 sense SEQ ID NO: 762 antisense SEQ ID NO: 763 sense SEQ ID NO: 764 antisense SEQ ID NO: 765 sense SEQ ID NO: 766 antisense SEQ ID NO: 767 sense SEQ ID NO: 768 antisense SEQ ID NO: 769 sense SEQ ID NO: 770 antisense SEQ ID NO: 771 sense SEQ ID NO: 772 antisense SEQ ID NO: 773 sense SEQ ID NO: 774 antisense SEQ ID NO: 775 sense SEQ ID NO: 776 antisense SEQ ID NO: 777 sense SEQ ID NO: 778 antisense SEQ ID NO: 779 sense SEQ ID NO: 780 antisense SEQ ID NO: 781 sense SEQ ID NO: 782 antisense SEQ ID NO: 783 sense SEQ ID NO: 784 antisense SEQ ID NO: 785 sense SEQ ID NO: 786 antisense SEQ ID NO: 787 sense SEQ ID NO: 788 antisense SEQ ID NO: 789 sense SEQ ID NO: 790 antisense SEQ ID NO: 791 sense SEQ ID NO: 792 antisense SEQ ID NO: 793 sense SEQ ID NO: 794 antisense SEQ ID NO: 795 sense SEQ ID NO: 796 antisense SEQ ID NO: 797 sense SEQ ID NO: 798 antisense SEQ ID NO: 799 sense SEQ ID NO: 800 antisense SEQ ID NO: 801 sense SEQ ID NO: 802 antisense SEQ ID NO: 803 sense SEQ ID NO: 804 antisense SEQ ID NO: 805 sense SEQ ID NO: 806 antisense SEQ ID NO: 807 sense SEQ ID NO: 808 antisense SEQ ID NO: 809 sense SEQ ID NO: 810 antisense SEQ ID NO: 811 sense SEQ ID NO: 812 antisense SEQ ID NO: 813 sense SEQ ID NO: 814 antisense SEQ ID NO: 815 sense SEQ ID NO: 816 antisense SEQ ID NO: 817 sense SEQ ID NO: 818 antisense SEQ ID NO: 819 sense SEQ ID NO: 820 antisense SEQ ID NO: 821 sense SEQ ID NO: 822 antisense SEQ ID NO: 823 sense SEQ ID NO: 824 antisense SEQ ID NO: 825 sense SEQ ID NO: 826 antisense SEQ ID NO: 827 sense SEQ ID NO: 828 antisense SEQ ID NO: 829 sense SEQ ID NO: 830 antisense SEQ ID NO: 831 sense SEQ ID NO: 832 antisense SEQ ID NO: 833 sense SEQ ID NO: 834 antisense SEQ ID NO: 835 sense SEQ ID NO: 836 antisense SEQ ID NO: 837 sense SEQ ID NO: 838 antisense SEQ ID NO: 839 sense SEQ ID NO: 840 antisense SEQ ID NO: 841 sense SEQ ID NO: 842 antisense SEQ ID NO: 843 sense SEQ ID NO: 844 antisense SEQ ID NO: 845 sense SEQ ID NO: 846 antisense SEQ ID NO: 847 sense SEQ ID NO: 848 antisense SEQ ID NO: 849 sense SEQ ID NO: 850 antisense SEQ ID NO: 851 sense SEQ ID NO: 852 antisense SEQ ID NO: 853 sense SEQ ID NO: 854 antisense SEQ ID NO: 855 sense SEQ ID NO: 856 antisense SEQ ID NO: 857 sense SEQ ID NO: 858 antisense SEQ ID NO: 859 sense SEQ ID NO: 860 antisense SEQ ID NO: 861 sense SEQ ID NO: 862 antisense SEQ ID NO: 863 sense SEQ ID NO: 864 antisense SEQ ID NO: 865 sense SEQ ID NO: 866 antisense SEQ ID NO: 867 sense SEQ ID NO: 868 antisense SEQ ID NO: 869 sense SEQ ID NO: 870 antisense SEQ ID NO: 871 sense SEQ ID NO: 872 antisense SEQ ID NO: 873 sense SEQ ID NO: 874 antisense SEQ ID NO: 875 sense SEQ ID NO: 876 antisense SEQ ID NO: 877 sense SEQ ID NO: 878 antisense SEQ ID NO: 879 sense SEQ ID NO: 880 antisense SEQ ID NO: 881 sense SEQ ID NO: 882 antisense SEQ ID NO: 883 sense SEQ ID NO: 884 antisense SEQ ID NO: 885 sense SEQ ID NO: 886 antisense SEQ ID NO: 887 sense SEQ ID NO: 888 antisense SEQ ID NO: 889 sense SEQ ID NO: 890 antisense SEQ ID NO: 891 sense SEQ ID NO: 892 antisense SEQ ID NO: 893 sense SEQ ID NO: 894 antisense SEQ ID NO: 895 sense SEQ ID NO: 896 antisense SEQ ID NO: 897 sense SEQ ID NO: 898 antisense SEQ ID NO: 899 sense SEQ ID NO: 900 antisense SEQ ID NO: 901 sense SEQ ID NO: 902 antisense SEQ ID NO: 903 sense SEQ ID NO: 904 antisense SEQ ID NO: 905 sense SEQ ID NO: 906 antisense SEQ ID NO: 907 sense SEQ ID NO: 908 antisense SEQ ID NO: 909 sense SEQ ID NO: 910 antisense SEQ ID NO: 911 sense SEQ ID NO: 912 antisense SEQ ID NO: 913 sense SEQ ID NO: 914 antisense SEQ ID NO: 915 sense SEQ ID NO: 916 antisense SEQ ID NO: 917 sense SEQ ID NO: 918 antisense SEQ ID NO: 919 sense SEQ ID NO: 920 antisense SEQ ID NO: 921 sense SEQ ID NO: 922 antisense SEQ ID NO: 923 sense SEQ ID NO: 924 antisense SEQ ID NO: 925 sense SEQ ID NO: 926 antisense SEQ ID NO: 927 sense SEQ ID NO: 928 antisense SEQ ID NO: 929 sense SEQ ID NO: 930 antisense SEQ ID NO: 931 sense SEQ ID NO: 932 antisense SEQ ID NO: 933 sense SEQ ID NO: 934 antisense SEQ ID NO: 935 sense SEQ ID NO: 936 antisense SEQ ID NO: 937 sense SEQ ID NO: 938 antisense SEQ ID NO: 939 sense SEQ ID NO: 940 antisense SEQ ID NO: 941 sense SEQ ID NO: 942 antisense SEQ ID NO: 943 sense SEQ ID NO: 944 antisense SEQ ID NO: 945 sense SEQ ID NO: 946 antisense SEQ ID NO: 947 sense SEQ ID NO: 948 antisense SEQ ID NO: 949 sense SEQ ID NO: 950 antisense SEQ ID NO: 951 sense SEQ ID NO: 952 antisense SEQ ID NO: 953 sense SEQ ID NO: 954 antisense SEQ ID NO: 955 sense SEQ ID NO: 956 antisense SEQ ID NO: 957 sense SEQ ID NO: 958 antisense SEQ ID NO: 959 sense SEQ ID NO: 960 antisense SEQ ID NO: 961 sense SEQ ID NO: 962 antisense SEQ ID NO: 963 sense SEQ ID NO: 964 antisense SEQ ID NO: 965 sense SEQ ID NO: 966 antisense SEQ ID NO: 967 sense SEQ ID NO: 968 antisense SEQ ID NO: 969 sense SEQ ID NO: 970 antisense SEQ ID NO: 971 sense SEQ ID NO: 972 antisense SEQ ID NO: 973 sense SEQ ID NO: 974 antisense SEQ ID NO: 975 sense SEQ ID NO: 976 antisense SEQ ID NO: 977 sense SEQ ID NO: 978 antisense SEQ ID NO: 979 sense SEQ ID NO: 980 antisense SEQ ID NO: 981 sense SEQ ID NO: 982 antisense SEQ ID NO: 983 sense SEQ ID NO: 984 antisense SEQ ID NO: 985 sense SEQ ID NO: 986 antisense SEQ ID NO: 987 sense SEQ ID NO: 988 antisense SEQ ID NO: 989 sense SEQ ID NO: 990 antisense SEQ ID NO: 991 sense SEQ ID NO: 992 antisense SEQ ID NO: 993 sense SEQ ID NO: 994 antisense SEQ ID NO: 995 sense SEQ ID NO: 996 antisense SEQ ID NO: 997 sense SEQ ID NO: 998 antisense SEQ ID NO: 999 sense SEQ ID NO: 1000 antisense SEQ ID NO: 1001 sense SEQ ID NO: 1002 antisense SEQ ID NO: 1003 sense SEQ ID NO: 1004 antisense SEQ ID NO: 1005 sense SEQ ID NO: 1006 antisense SEQ ID NO: 1007 sense SEQ ID NO: 1008 antisense SEQ ID NO: 1009 sense SEQ ID NO: 1010 antisense SEQ ID NO: 1011 sense SEQ ID NO: 1012 antisense SEQ ID NO: 1013 sense SEQ ID NO: 1014 antisense SEQ ID NO: 1015 sense SEQ ID NO: 1016 antisense SEQ ID NO: 1017 sense SEQ ID NO: 1018 antisense SEQ ID NO: 1019 sense SEQ ID NO: 1020 antisense SEQ ID NO: 1021 sense SEQ ID NO: 1022 antisense SEQ ID NO: 1023 sense SEQ ID NO: 1024 antisense SEQ ID NO: 1025 sense SEQ ID NO: 1026 antisense SEQ ID NO: 1027 sense SEQ ID NO: 1028 antisense SEQ ID NO: 1029 sense SEQ ID NO: 1030 antisense SEQ ID NO: 1031 sense SEQ ID NO: 1032 antisense SEQ ID NO: 1033 sense SEQ ID NO: 1034 antisense SEQ ID NO: 1035 sense SEQ ID NO: 1036 antisense SEQ ID NO: 1037 sense SEQ ID NO: 1038 antisense SEQ ID NO: 1039 sense SEQ ID NO: 1040 antisense SEQ ID NO: 1041 sense SEQ ID NO: 1042 antisense SEQ ID NO: 1043 sense SEQ ID NO: 1044 antisense SEQ ID NO: 1045 sense SEQ ID NO: 1046 antisense SEQ ID NO: 1047 sense SEQ ID NO: 1048 antisense SEQ ID NO: 1049 sense SEQ ID NO: 1050 antisense SEQ ID NO: 1051 sense SEQ ID NO: 1052 antisense SEQ ID NO: 1053 sense SEQ ID NO: 1054 antisense SEQ ID NO: 1055 sense SEQ ID NO: 1056 antisense SEQ ID NO: 1057 sense SEQ ID NO: 1058 antisense SEQ ID NO: 1059 sense SEQ ID NO: 1060 antisense SEQ ID NO: 1061 sense SEQ ID NO: 1062 antisense SEQ ID NO: 1063 sense SEQ ID NO: 1064 antisense SEQ ID NO: 1065 sense SEQ ID NO: 1066 antisense SEQ ID NO: 1067 sense SEQ ID NO: 1068 antisense SEQ ID NO: 1069 sense SEQ ID NO: 1070 antisense SEQ ID NO: 1071 sense SEQ ID NO: 1072 antisense SEQ ID NO: 1073 sense SEQ ID NO: 1074 antisense SEQ ID NO: 1075 sense SEQ ID NO: 1076 antisense SEQ ID NO: 1077 sense SEQ ID NO: 1078 antisense SEQ ID NO: 1079 sense SEQ ID NO: 1080 antisense SEQ ID NO: 1081 sense SEQ ID NO: 1082 antisense SEQ ID NO: 1083 sense SEQ ID NO: 1084 antisense SEQ ID NO: 1085 sense SEQ ID NO: 1086 antisense SEQ ID NO: 1087 sense SEQ ID NO: 1088 antisense SEQ ID NO: 1089 sense SEQ ID NO: 1090 antisense SEQ ID NO: 1091 sense SEQ ID NO: 1092 antisense SEQ ID NO: 1093 sense SEQ ID NO: 1094 antisense SEQ ID NO: 1095 sense SEQ ID NO: 1096 antisense SEQ ID NO: 1097 sense SEQ ID NO: 1098 antisense SEQ ID NO: 1099 sense SEQ ID NO: 1100 antisense SEQ ID NO: 1101 sense SEQ ID NO: 1102 antisense SEQ ID NO: 1103 sense SEQ ID NO: 1104 antisense SEQ ID NO: 1105 sense SEQ ID NO: 1106 antisense SEQ ID NO: 1107 sense SEQ ID NO: 1108 antisense SEQ ID NO: 1109 sense SEQ ID NO: 1110 antisense SEQ ID NO: 1111 sense SEQ ID NO: 1112 antisense SEQ ID NO: 1113 sense SEQ ID NO: 1114 antisense SEQ ID NO: 1115 sense SEQ ID NO: 1116 antisense SEQ ID NO: 1117 sense SEQ ID NO: 1118 antisense SEQ ID NO: 1119 sense SEQ ID NO: 1120 antisense SEQ ID NO: 1121 sense SEQ ID NO: 1122 antisense SEQ ID NO: 1123 sense SEQ ID NO: 1124 antisense SEQ ID NO: 1125 sense SEQ ID NO: 1126 antisense SEQ ID NO: 1127 sense SEQ ID NO: 1128 antisense SEQ ID NO: 1129 sense SEQ ID NO: 1130 antisense SEQ ID NO: 1131 sense SEQ ID NO: 1132 antisense SEQ ID NO: 1133 sense SEQ ID NO: 1134 antisense SEQ ID NO: 1135 sense SEQ ID NO: 1136 antisense SEQ ID NO: 1137 sense SEQ ID NO: 1138 antisense SEQ ID NO: 1139 sense SEQ ID NO: 1140 antisense SEQ ID NO: 1141 sense SEQ ID NO: 1142 antisense SEQ ID NO: 1143 sense SEQ ID NO: 1144 antisense SEQ ID NO: 1145 sense SEQ ID NO: 1146 antisense SEQ ID NO: 1147 sense SEQ ID NO: 1148 antisense SEQ ID NO: 1149 sense SEQ ID NO: 1150 antisense SEQ ID NO: 1151 sense SEQ ID NO: 1152 antisense SEQ ID NO: 1153 sense SEQ ID NO: 1154 antisense SEQ ID NO: 1155 sense SEQ ID NO: 1156 antisense SEQ ID NO: 1157 sense SEQ ID NO: 1158 antisense SEQ ID NO: 1159 sense SEQ ID NO: 1160 antisense SEQ ID NO: 1161 sense SEQ ID NO: 1162 antisense SEQ ID NO: 1163 sense SEQ ID NO: 1164 antisense SEQ ID NO: 1165 sense SEQ ID NO: 1166 antisense SEQ ID NO: 1167 sense SEQ ID NO: 1168 antisense SEQ ID NO: 1169 sense SEQ ID NO: 1170 antisense SEQ ID NO: 1171 sense SEQ ID NO: 1172 antisense SEQ ID NO: 1173 sense SEQ ID NO: 1174 antisense SEQ ID NO: 1175 sense SEQ ID NO: 1176 antisense SEQ ID NO: 1177 sense SEQ ID NO: 1178 antisense SEQ ID NO: 1179 sense SEQ ID NO: 1180 antisense SEQ ID NO: 1181 sense SEQ ID NO: 1182 antisense SEQ ID NO: 1183 sense SEQ ID NO: 1184 antisense SEQ ID NO: 1185 sense SEQ ID NO: 1186 antisense SEQ ID NO: 1187 sense SEQ ID NO: 1188 antisense SEQ ID NO: 1189 sense SEQ ID NO: 1190 antisense SEQ ID NO: 1191 sense SEQ ID NO: 1192 antisense SEQ ID NO: 1193 sense SEQ ID NO: 1194 antisense SEQ ID NO: 1195 sense SEQ ID NO: 1196 antisense SEQ ID NO: 1197 sense SEQ ID NO: 1198 antisense SEQ ID NO: 1199 sense SEQ ID NO: 1200 antisense SEQ ID NO: 1201 sense SEQ ID NO: 1202 antisense SEQ ID NO: 1203 sense SEQ ID NO: 1204 antisense SEQ ID NO: 1205 sense SEQ ID NO: 1206 antisense SEQ ID NO: 1207 sense SEQ ID NO: 1208 antisense SEQ ID NO: 1209 sense SEQ ID NO: 1210 antisense SEQ ID NO: 1211 sense SEQ ID NO: 1212 antisense SEQ ID NO: 1213 sense SEQ ID NO: 1214 antisense SEQ ID NO: 1215 sense SEQ ID NO: 1216 antisense SEQ ID NO: 1217 sense SEQ ID NO: 1218 antisense SEQ ID NO: 1219 sense SEQ ID NO: 1220 antisense SEQ ID NO: 1221 sense SEQ ID NO: 1222 antisense SEQ ID NO: 1223 sense SEQ ID NO: 1224 antisense SEQ ID NO: 1225 sense SEQ ID NO: 1226 antisense SEQ ID NO: 1227 sense SEQ ID NO: 1228 antisense SEQ ID NO: 1229 sense SEQ ID NO: 1230 antisense SEQ ID NO: 1231 sense SEQ ID NO: 1232 antisense SEQ ID NO: 1233 sense SEQ ID NO: 1234 antisense SEQ ID NO: 1235 sense SEQ ID NO: 1236 antisense SEQ ID NO: 1237 sense SEQ ID NO: 1238 antisense SEQ ID NO: 1239 sense SEQ ID NO: 1240 antisense SEQ ID NO: 1241 sense SEQ ID NO: 1242 antisense SEQ ID NO: 1243 sense SEQ ID NO: 1244 antisense SEQ ID NO: 1245 sense SEQ ID NO: 1246 antisense SEQ ID NO: 1247 sense SEQ ID NO: 1248 antisense SEQ ID NO: 1249 sense SEQ ID NO: 1250 antisense SEQ ID NO: 1251 sense SEQ ID NO: 1252 antisense SEQ ID NO: 1253 sense SEQ ID NO: 1254 antisense SEQ ID NO: 1255 sense SEQ ID NO: 1256 antisense SEQ ID NO: 1257 sense SEQ ID NO: 1258 antisense SEQ ID NO: 1259 sense SEQ ID NO: 1260 antisense SEQ ID NO: 1261 sense SEQ ID NO: 1262 antisense SEQ ID NO: 1263 sense SEQ ID NO: 1264 antisense SEQ ID NO: 1265 sense SEQ ID NO: 1266 antisense SEQ ID NO: 1267 sense SEQ ID NO: 1268 antisense SEQ ID NO: 1269 sense SEQ ID NO: 1270 antisense SEQ ID NO: 1271 sense SEQ ID NO: 1272 antisense SEQ ID NO: 1273 sense SEQ ID NO: 1274 antisense SEQ ID NO: 1275 sense SEQ ID NO: 1276 antisense SEQ ID NO: 1277 sense SEQ ID NO: 1278 antisense SEQ ID NO: 1279 sense SEQ ID NO: 1280 antisense SEQ ID NO: 1281 sense SEQ ID NO: 1282 antisense SEQ ID NO: 1283 sense SEQ ID NO: 1284 antisense SEQ ID NO: 1285 sense SEQ ID NO: 1286 antisense SEQ ID NO: 1287 sense SEQ ID NO: 1288 antisense SEQ ID NO: 1289 sense SEQ ID NO: 1290 antisense SEQ ID NO: 1291 sense SEQ ID NO: 1292 antisense SEQ ID NO: 1293 sense SEQ ID NO: 1294 antisense SEQ ID NO: 1295 sense SEQ ID NO: 1296 antisense SEQ ID NO: 1297 sense SEQ ID NO: 1298 antisense SEQ ID NO: 1299 sense SEQ ID NO: 1300 antisense SEQ ID NO: 1301 sense SEQ ID NO: 1302 antisense SEQ ID NO: 1303 sense SEQ ID NO: 1304 antisense SEQ ID NO: 1305 sense SEQ ID NO: 1306 antisense SEQ ID NO: 1307 sense SEQ ID NO: 1308 antisense SEQ ID NO: 1309 sense SEQ ID NO: 1310 antisense SEQ ID NO: 1311 sense SEQ ID NO: 1312 antisense SEQ ID NO: 1313 sense SEQ ID NO: 1314 antisense SEQ ID NO: 1315 sense SEQ ID NO: 1316 antisense SEQ ID NO: 1317 sense SEQ ID NO: 1318 antisense SEQ ID NO: 1319 sense SEQ ID NO: 1320 antisense SEQ ID NO: 1321 sense SEQ ID NO: 1322 antisense SEQ ID NO: 1323 sense SEQ ID NO: 1324 antisense SEQ ID NO: 1325 sense SEQ ID NO: 1326 antisense SEQ ID NO: 1327 sense SEQ ID NO: 1328 antisense SEQ ID NO: 1329 sense SEQ ID NO: 1330 antisense SEQ ID NO: 1331 sense SEQ ID NO: 1332 antisense SEQ ID NO: 1333 sense SEQ ID NO: 1334 antisense SEQ ID NO: 1335 sense SEQ ID NO: 1336 antisense SEQ ID NO: 1337 sense SEQ ID NO: 1338 antisense SEQ ID NO: 1339 sense SEQ ID NO: 1340 antisense SEQ ID NO: 1341 sense SEQ ID NO: 1342 antisense SEQ ID NO: 1343 sense SEQ ID NO: 1344 antisense SEQ ID NO: 1345 sense SEQ ID NO: 1346 antisense SEQ ID NO: 1347 sense SEQ ID NO: 1348 antisense SEQ ID NO: 1349 sense SEQ ID NO: 1350 antisense SEQ ID NO: 1351 sense SEQ ID NO: 1352 antisense SEQ ID NO: 1353 sense SEQ ID NO: 1354 antisense SEQ ID NO: 1355 sense SEQ ID NO: 1356 antisense SEQ ID NO: 1357 sense SEQ ID NO: 1358 antisense SEQ ID NO: 1359 sense SEQ ID NO: 1360 antisense SEQ ID NO: 1361 sense SEQ ID NO: 1362 antisense SEQ ID NO: 1363 sense SEQ ID NO: 1364 antisense SEQ ID NO: 1365 sense SEQ ID NO: 1366 antisense SEQ ID NO: 1367 sense SEQ ID NO: 1368 antisense SEQ ID NO: 1369 sense SEQ ID NO: 1370 antisense SEQ ID NO: 1371 sense SEQ ID NO: 1372 antisense SEQ ID NO: 1373 sense SEQ ID NO: 1374 antisense SEQ ID NO: 1375 sense SEQ ID NO: 1376 antisense SEQ ID NO: 1377 sense SEQ ID NO: 1378 antisense SEQ ID NO: 1379 sense SEQ ID NO: 1380 antisense SEQ ID NO: 1381 sense SEQ ID NO: 1382 antisense SEQ ID NO: 1383 sense SEQ ID NO: 1384 antisense SEQ ID NO: 1385 sense SEQ ID NO: 1386 antisense SEQ ID NO: 1387 sense SEQ ID NO: 1388 antisense SEQ ID NO: 1389 sense SEQ ID NO: 1390 antisense SEQ ID NO: 1391 sense SEQ ID NO: 1392 antisense SEQ ID NO: 1393 sense SEQ ID NO: 1394 antisense SEQ ID NO: 1395 sense SEQ ID NO: 1396 antisense SEQ ID NO: 1397 sense SEQ ID NO: 1398 antisense SEQ ID NO: 1399 sense SEQ ID NO: 1400 antisense SEQ ID NO: 1401 sense SEQ ID NO: 1402 antisense SEQ ID NO: 1403 sense SEQ ID NO: 1404 antisense SEQ ID NO: 1405 sense SEQ ID NO: 1406 antisense SEQ ID NO: 1407 sense SEQ ID NO: 1408 antisense SEQ ID NO: 1409 sense SEQ ID NO: 1410 antisense SEQ ID NO: 1411 sense SEQ ID NO: 1412 antisense SEQ ID NO: 1413 sense SEQ ID NO: 1414 antisense SEQ ID NO: 1415 sense SEQ ID NO: 1416 antisense SEQ ID NO: 1417 sense SEQ ID NO: 1418 antisense SEQ ID NO: 1419 sense SEQ ID NO: 1420 antisense SEQ ID NO: 1421 sense SEQ ID NO: 1422 antisense SEQ ID NO: 1423 sense SEQ ID NO: 1424 antisense SEQ ID NO: 1425 sense SEQ ID NO: 1426 antisense SEQ ID NO: 1427 sense SEQ ID NO: 1428 antisense SEQ ID NO: 1429 sense SEQ ID NO: 1430 antisense SEQ ID NO: 1431 sense SEQ ID NO: 1432 antisense SEQ ID NO: 1433 sense SEQ ID NO: 1434 antisense SEQ ID NO: 1435 sense SEQ ID NO: 1436 antisense SEQ ID NO: 1437 sense SEQ ID NO: 1438 antisense SEQ ID NO: 1439 sense SEQ ID NO: 1440 antisense SEQ ID NO: 1441 sense SEQ ID NO: 1442 antisense SEQ ID NO: 1443 sense SEQ ID NO: 1444 antisense SEQ ID NO: 1445 sense SEQ ID NO: 1446 antisense SEQ ID NO: 1447 sense SEQ ID NO: 1448 antisense SEQ ID NO: 1449 sense SEQ ID NO: 1450 antisense SEQ ID NO: 1451 sense SEQ ID NO: 1452 antisense SEQ ID NO: 1453 sense SEQ ID NO: 1454 antisense SEQ ID NO: 1455 sense SEQ ID NO: 1456 antisense SEQ ID NO: 1457 sense SEQ ID NO: 1458 antisense SEQ ID NO: 1459 sense SEQ ID NO: 1460 antisense SEQ ID NO: 1461 sense SEQ ID NO: 1462 antisense SEQ ID NO: 1463 sense SEQ ID NO: 1464 antisense SEQ ID NO: 1465 sense SEQ ID NO: 1466 antisense SEQ ID NO: 1467 sense SEQ ID NO: 1468 antisense SEQ ID NO: 1469 sense SEQ ID NO: 1470 antisense SEQ ID NO: 1471 sense SEQ ID NO: 1472 antisense SEQ ID NO: 1473 sense SEQ ID NO: 1474 antisense SEQ ID NO: 1475 sense SEQ ID NO: 1476 antisense SEQ ID NO: 1477 sense SEQ ID NO: 1478 antisense SEQ ID NO: 1479 sense SEQ ID NO: 1480 antisense SEQ ID NO: 1481 sense SEQ ID NO: 1482 antisense SEQ ID NO: 1483 sense SEQ ID NO: 1484 antisense SEQ ID NO: 1485 sense SEQ ID NO: 1486 antisense SEQ ID NO: 1487 sense SEQ ID NO: 1488 antisense SEQ ID NO: 1489 sense SEQ ID NO: 1490 antisense SEQ ID NO: 1491 sense SEQ ID NO: 1492 antisense SEQ ID NO: 1493 sense SEQ ID NO: 1494 antisense SEQ ID NO: 1495 sense SEQ ID NO: 1496 antisense SEQ ID NO: 1497 sense SEQ ID NO: 1498 antisense SEQ ID NO: 1499 sense SEQ ID NO: 1500 antisense SEQ ID NO: 1501 sense SEQ ID NO: 1502 antisense SEQ ID NO: 1503 sense SEQ ID NO: 1504 antisense SEQ ID NO: 1505 sense SEQ ID NO: 1506 antisense SEQ ID NO: 1507 sense SEQ ID NO: 1508 antisense SEQ ID NO: 1509 sense SEQ ID NO: 1510 antisense SEQ ID NO: 1511 sense SEQ ID NO: 1512 antisense SEQ ID NO: 1513 sense SEQ ID NO: 1514 antisense SEQ ID NO: 1515 sense SEQ ID NO: 1516 antisense SEQ ID NO: 1517 sense SEQ ID NO: 1518 antisense SEQ ID NO: 1519 sense SEQ ID NO: 1520 antisense SEQ ID NO: 1521 sense SEQ ID NO: 1522 antisense SEQ ID NO: 1523 sense SEQ ID NO: 1524 antisense SEQ ID NO: 1525 sense SEQ ID NO: 1526 antisense SEQ ID NO: 1527 sense SEQ ID NO: 1528 antisense SEQ ID NO: 1529 sense SEQ ID NO: 1530 antisense SEQ ID NO: 1531 sense SEQ ID NO: 1532 antisense SEQ ID NO: 1533 sense SEQ ID NO: 1534 antisense SEQ ID NO: 1535 sense SEQ ID NO: 1536 antisense SEQ ID NO: 1537 sense SEQ ID NO: 1538 antisense SEQ ID NO: 1539 sense SEQ ID NO: 1540 antisense SEQ ID NO: 1541 sense SEQ ID NO: 1542 antisense SEQ ID NO: 1543 sense SEQ ID NO: 1544 antisense SEQ ID NO: 1545 sense SEQ ID NO: 1546 antisense SEQ ID NO: 1547 sense SEQ ID NO: 1548 antisense SEQ ID NO: 1549 sense SEQ ID NO: 1550 antisense SEQ ID NO: 1551 sense SEQ ID NO: 1552 antisense SEQ ID NO: 1553 sense SEQ ID NO: 1554 antisense SEQ ID NO: 1555 sense SEQ ID NO: 1556 antisense SEQ ID NO: 1557 sense SEQ ID NO: 1558 antisense SEQ ID NO: 1559 sense SEQ ID NO: 1560 antisense SEQ ID NO: 1561 sense SEQ ID NO: 1562 antisense SEQ ID NO: 1563 sense SEQ ID NO: 1564 antisense SEQ ID NO: 1565 sense SEQ ID NO: 1566 antisense SEQ ID NO: 1567 sense SEQ ID NO: 1568 antisense SEQ ID NO: 1569 sense SEQ ID NO: 1570 antisense SEQ ID NO: 1571 sense SEQ ID NO: 1572 antisense SEQ ID NO: 1573 sense SEQ ID NO: 1574 antisense SEQ ID NO: 1575 sense SEQ ID NO: 1576 antisense SEQ ID NO: 1577 sense SEQ ID NO: 1578 antisense SEQ ID NO: 1579 sense SEQ ID NO: 1580 antisense SEQ ID NO: 1581 sense SEQ ID NO: 1582 antisense SEQ ID NO: 1583 sense SEQ ID NO: 1584 antisense SEQ ID NO: 1585 sense SEQ ID NO: 1586 antisense SEQ ID NO: 1587 sense SEQ ID NO: 1588 antisense SEQ ID NO: 1589 sense SEQ ID NO: 1590 antisense SEQ ID NO: 1591 sense SEQ ID NO: 1592 antisense SEQ ID NO: 1593 sense SEQ ID NO: 1594 antisense SEQ ID NO: 1595 sense SEQ ID NO: 1596 antisense SEQ ID NO: 1597 sense SEQ ID NO: 1598 antisense SEQ ID NO: 1599 sense SEQ ID NO: 1600 antisense SEQ ID NO: 1601 sense SEQ ID NO: 1602 antisense SEQ ID NO: 1603 sense SEQ ID NO: 1604 antisense SEQ ID NO: 1605 sense SEQ ID NO: 1606 antisense SEQ ID NO: 1607 sense SEQ ID NO: 1608 antisense SEQ ID NO: 1609 sense SEQ ID NO: 1610 antisense SEQ ID NO: 1611 sense SEQ ID NO: 1612 antisense SEQ ID NO: 1613 sense SEQ ID NO: 1614 antisense SEQ ID NO: 1615 sense SEQ ID NO: 1616 antisense SEQ ID NO: 1617 sense SEQ ID NO: 1618 antisense SEQ ID NO: 1619 sense SEQ ID NO: 1620 antisense SEQ ID NO: 1621 sense SEQ ID NO: 1622 antisense SEQ ID NO: 1623 sense SEQ ID NO: 1624 antisense SEQ ID NO: 1625 sense SEQ ID NO: 1626 antisense SEQ ID NO: 1627 sense SEQ ID NO: 1628 antisense SEQ ID NO: 1629 sense SEQ ID NO: 1630 antisense SEQ ID NO: 1631 sense SEQ ID NO: 1632 antisense SEQ ID NO: 1633 sense SEQ ID NO: 1634 antisense SEQ ID NO: 1635 sense SEQ ID NO: 1636 antisense SEQ ID NO: 1637 sense SEQ ID NO: 1638 antisense SEQ ID NO: 1639 sense SEQ ID NO: 1640 antisense SEQ ID NO: 1641 sense SEQ ID NO: 1642 antisense SEQ ID NO: 1643 sense SEQ ID NO: 1644 antisense SEQ ID NO: 1645 sense SEQ ID NO: 1646 antisense SEQ ID NO: 1647 sense SEQ ID NO: 1648 antisense SEQ ID NO: 1649 sense SEQ ID NO: 1650 antisense SEQ ID NO: 1651 sense SEQ ID NO: 1652 antisense SEQ ID NO: 1653 sense SEQ ID NO: 1654 antisense SEQ ID NO: 1655 sense SEQ ID NO: 1656 antisense SEQ ID NO: 1657 sense SEQ ID NO: 1658 antisense SEQ ID NO: 1659 sense SEQ ID NO: 1660 antisense SEQ ID NO: 1661 sense SEQ ID NO: 1662 antisense SEQ ID NO: 1663 sense SEQ ID NO: 1664 antisense SEQ ID NO: 1665 sense SEQ ID NO: 1666 antisense SEQ ID NO: 1667 sense SEQ ID NO: 1668 antisense SEQ ID NO: 1669 sense SEQ ID NO: 1670 antisense SEQ ID NO: 1671 sense SEQ ID NO: 1672 antisense SEQ ID NO: 1673 sense SEQ ID NO: 1674 antisense SEQ ID NO: 1675 sense SEQ ID NO: 1676 antisense SEQ ID NO: 1677 sense SEQ ID NO: 1678 antisense SEQ ID NO: 1679 sense SEQ ID NO: 1680 antisense SEQ ID NO: 1681 sense SEQ ID NO: 1682 antisense SEQ ID NO: 1683 sense SEQ ID NO: 1684 antisense SEQ ID NO: 1685 sense SEQ ID NO: 1686 antisense SEQ ID NO: 1687 sense SEQ ID NO: 1688 antisense SEQ ID NO: 1689 sense SEQ ID NO: 1690 antisense SEQ ID NO: 1691 sense SEQ ID NO: 1692 antisense SEQ ID NO: 1693 sense SEQ ID NO: 1694 antisense SEQ ID NO: 1695 sense SEQ ID NO: 1696 antisense SEQ ID NO: 1697 sense SEQ ID NO: 1698 antisense SEQ ID NO: 1699 sense SEQ ID NO: 1700 antisense SEQ ID NO: 1701 sense SEQ ID NO: 1702 antisense SEQ ID NO: 1703 sense SEQ ID NO: 1704 antisense SEQ ID NO: 1705 sense SEQ ID NO: 1706 antisense SEQ ID NO: 1707 sense SEQ ID NO: 1708 antisense SEQ ID NO: 1709 sense SEQ ID NO: 1710 antisense SEQ ID NO: 1711 sense SEQ ID NO: 1712 antisense SEQ ID NO: 1713 sense SEQ ID NO: 1714 antisense SEQ ID NO: 1715 sense SEQ ID NO: 1716 antisense SEQ ID NO: 1717 sense SEQ ID NO: 1718 antisense SEQ ID NO: 1719 sense SEQ ID NO: 1720 antisense SEQ ID NO: 1721 sense SEQ ID NO: 1722 antisense SEQ ID NO: 1723 sense SEQ ID NO: 1724 antisense SEQ ID NO: 1725 sense SEQ ID NO: 1726 antisense SEQ ID NO: 1727 sense SEQ ID NO: 1728 antisense SEQ ID NO: 1729 sense SEQ ID NO: 1730 antisense SEQ ID NO: 1731 sense SEQ ID NO: 1732 antisense SEQ ID NO: 1733 sense SEQ ID NO: 1734 antisense SEQ ID NO: 1735 sense SEQ ID NO: 1736 antisense SEQ ID NO: 1737 sense SEQ ID NO: 1738 antisense SEQ ID NO: 1739 sense SEQ ID NO: 1740 antisense SEQ ID NO: 1741 sense SEQ ID NO: 1742 antisense SEQ ID NO: 1743 sense SEQ ID NO: 1744 antisense SEQ ID NO: 1745 sense SEQ ID NO: 1746 antisense SEQ ID NO: 1747 sense SEQ ID NO: 1748 antisense SEQ ID NO: 1749 sense SEQ ID NO: 1750 antisense SEQ ID NO: 1751 sense SEQ ID NO: 1752 antisense SEQ ID NO: 1753 sense SEQ ID NO: 1754 antisense SEQ ID NO: 1755 sense SEQ ID NO: 1756 antisense SEQ ID NO: 1757 sense SEQ ID NO: 1758 antisense SEQ ID NO: 1759 sense SEQ ID NO: 1760 antisense SEQ ID NO: 1761 sense SEQ ID NO: 1760 antisense SEQ ID NO: 1763 sense SEQ ID NO: 1764 antisense SEQ ID NO: 1765 sense SEQ ID NO: 1764 antisense SEQ ID NO: 1767 sense SEQ ID NO: 1768 antisense SEQ ID NO: 1769 sense SEQ ID NO: 1770 antisense SEQ ID NO: 1771 sense SEQ ID NO: 1772 antisense SEQ ID NO: 1773 sense SEQ ID NO: 1774 antisense SEQ ID NO: 1775 sense SEQ ID NO: 1776 antisense SEQ ID NO: 1777 sense SEQ ID NO: 1778 antisense SEQ ID NO: 1779 sense SEQ ID NO: 1780 antisense SEQ ID NO: 1781 sense SEQ ID NO: 1782 antisense SEQ ID NO: 1783 sense SEQ ID NO: 1784 antisense SEQ ID NO: 1785 sense SEQ ID NO: 1786 antisense SEQ ID NO: 1787 sense SEQ ID NO: 1788 antisense SEQ ID NO: 1789 sense SEQ ID NO: 1790 antisense SEQ ID NO: 1791 sense SEQ ID NO: 1792 antisense SEQ ID NO: 1793 sense SEQ ID NO: 1794 antisense SEQ ID NO: 1795 sense SEQ ID NO: 1796 antisense SEQ ID NO: 1797 sense SEQ ID NO: 1798 antisense SEQ ID NO: 1799 sense SEQ ID NO: 1800 antisense SEQ ID NO: 1801 sense SEQ ID NO: 1802 antisense SEQ ID NO: 1803 sense SEQ ID NO: 1804 antisense SEQ ID NO: 1805 sense SEQ ID NO: 1806 antisense SEQ ID NO: 1807 sense SEQ ID NO: 1808 antisense SEQ ID NO: 1809 sense SEQ ID NO: 1810 antisense SEQ ID NO: 1811 sense SEQ ID NO: 1812 antisense SEQ ID NO: 1813 sense SEQ ID NO: 1814 antisense SEQ ID NO: 1815 sense SEQ ID NO: 1816 antisense SEQ ID NO: 1817 sense SEQ ID NO: 1818 antisense SEQ ID NO: 1819 sense SEQ ID NO: 1820 antisense SEQ ID NO: 1821 sense SEQ ID NO: 1822 antisense SEQ ID NO: 1823 sense SEQ ID NO: 1824 antisense SEQ ID NO: 1825 sense SEQ ID NO: 1826 antisense SEQ ID NO: 1827 sense SEQ ID NO: 1828 antisense SEQ ID NO: 1829 sense SEQ ID NO: 1830 antisense SEQ ID NO: 1831 sense SEQ ID NO: 1832 antisense SEQ ID NO: 1833 sense SEQ ID NO: 1834 antisense SEQ ID NO: 1835 sense SEQ ID NO: 1836 antisense SEQ ID NO: 1837 sense SEQ ID NO: 1838 antisense SEQ ID NO: 1839 sense SEQ ID NO: 1840 antisense SEQ ID NO: 1841 sense SEQ ID NO: 1842 antisense SEQ ID NO: 1843 sense SEQ ID NO: 1844 antisense SEQ ID NO: 1845 sense SEQ ID NO: 1846 antisense SEQ ID NO: 1847 sense SEQ ID NO: 1848 antisense SEQ ID NO: 1849 sense SEQ ID NO: 1850 antisense SEQ ID NO: 1851 sense SEQ ID NO: 1852 antisense SEQ ID NO: 1853 sense SEQ ID NO: 1854 antisense SEQ ID NO: 1855 sense SEQ ID NO: 1856 antisense SEQ ID NO: 1857 sense SEQ ID NO: 1858 antisense SEQ ID NO: 1859 sense SEQ ID NO: 1860 antisense SEQ ID NO: 1861 sense SEQ ID NO: 1862 antisense SEQ ID NO: 1863 sense SEQ ID NO: 1864 antisense SEQ ID NO: 1865 sense SEQ ID NO: 1866 antisense SEQ ID NO: 1867 sense SEQ ID NO: 1868 antisense SEQ ID NO: 1869 sense SEQ ID NO: 1870 antisense SEQ ID NO: 1871 sense SEQ ID NO: 1872 antisense SEQ ID NO: 1873 sense SEQ ID NO: 1874 antisense SEQ ID NO: 1875 sense SEQ ID NO: 1876 antisense SEQ ID NO: 1877 sense SEQ ID NO: 1878 antisense SEQ ID NO: 1879 sense SEQ ID NO: 1880 antisense SEQ ID NO: 1881 sense SEQ ID NO: 1882 antisense SEQ ID NO: 1883 sense SEQ ID NO: 1884 antisense SEQ ID NO: 1885 sense SEQ ID NO: 1886 antisense SEQ ID NO: 1887 sense SEQ ID NO: 1888 antisense SEQ ID NO: 1889 sense SEQ ID NO: 1890 antisense SEQ ID NO: 1891 sense SEQ ID NO: 1892 antisense SEQ ID NO: 1893 sense SEQ ID NO: 1894 antisense SEQ ID NO: 1895 sense SEQ ID NO: 1896 antisense SEQ ID NO: 1897 sense SEQ ID NO: 1898 antisense SEQ ID NO: 1899 sense SEQ ID NO: 1900 antisense SEQ ID NO: 1901 sense SEQ ID NO: 1902 antisense SEQ ID NO: 1903 sense SEQ ID NO: 1904 antisense SEQ ID NO: 1905 sense SEQ ID NO: 1906 antisense SEQ ID NO: 1907 sense SEQ ID NO: 1908 antisense SEQ ID NO: 1909 sense SEQ ID NO: 1910 antisense SEQ ID NO: 1911 sense SEQ ID NO: 1912 antisense SEQ ID NO: 1913 sense SEQ ID NO: 1914 antisense SEQ ID NO: 1915 sense SEQ ID NO: 1916 antisense SEQ ID NO: 1917 sense SEQ ID NO: 1918 antisense SEQ ID NO: 1919 sense SEQ ID NO: 1920 antisense SEQ ID NO: 1921 sense SEQ ID NO: 1922 antisense SEQ ID NO: 1923 sense SEQ ID NO: 1924 antisense SEQ ID NO: 1925 sense SEQ ID NO: 1926 antisense SEQ ID NO: 1927 sense SEQ ID NO: 1928 antisense SEQ ID NO: 1929 sense SEQ ID NO: 1930 antisense SEQ ID NO: 1931 sense SEQ ID NO: 1932 antisense SEQ ID NO: 1933 sense SEQ ID NO: 1934 antisense SEQ ID NO: 1935 sense SEQ ID NO: 1936 antisense SEQ ID NO: 1937 sense SEQ ID NO: 1938 antisense SEQ ID NO: 1939 sense SEQ ID NO: 1940 antisense SEQ ID NO: 1941 sense SEQ ID NO: 1942 antisense SEQ ID NO: 1943 sense SEQ ID NO: 1944 antisense SEQ ID NO: 1945 sense SEQ ID NO: 1946 antisense SEQ ID NO: 1947 sense SEQ ID NO: 1948 antisense SEQ ID NO: 1949 sense SEQ ID NO: 1950 antisense SEQ ID NO: 1951 sense SEQ ID NO: 1952 antisense SEQ ID NO: 1953 sense SEQ ID NO: 1954 antisense SEQ ID NO: 1955 sense SEQ ID NO: 1956 antisense SEQ ID NO: 1957 sense SEQ ID NO: 1958 antisense SEQ ID NO: 1959 sense SEQ ID NO: 1960 antisense SEQ ID NO: 1961 sense SEQ ID NO: 1962 antisense SEQ ID NO: 1963 sense SEQ ID NO: 1964 antisense SEQ ID NO: 1965 sense SEQ ID NO: 1966 antisense SEQ ID NO: 1967 sense SEQ ID NO: 1968 antisense SEQ ID NO: 1969 sense SEQ ID NO: 1970 antisense SEQ ID NO: 1971 sense SEQ ID NO: 1972 antisense SEQ ID NO: 1973 sense SEQ ID NO: 1974 antisense SEQ ID NO: 1975 sense SEQ ID NO: 1976 antisense SEQ ID NO: 1977 sense SEQ ID NO: 1978 antisense SEQ ID NO: 1979 sense SEQ ID NO: 1980 antisense SEQ ID NO: 1981 sense SEQ ID NO: 1982 antisense SEQ ID NO: 1983 sense SEQ ID NO: 1984 antisense SEQ ID NO: 1985 sense SEQ ID NO: 1986 antisense SEQ ID NO: 1987 sense SEQ ID NO: 1988 antisense SEQ ID NO: 1989 sense SEQ ID NO: 1990 antisense SEQ ID NO: 1991 sense SEQ ID NO: 1992 antisense SEQ ID NO: 1993 sense SEQ ID NO: 1994 antisense SEQ ID NO: 1995 sense SEQ ID NO: 1996 antisense SEQ ID NO: 1997 sense SEQ ID NO: 1998 antisense SEQ ID NO: 1999 sense SEQ ID NO: 2000 antisense SEQ ID NO: 2001 sense SEQ ID NO: 2002 antisense SEQ ID NO: 2003 sense SEQ ID NO: 2004 antisense SEQ ID NO: 2005 sense SEQ ID NO: 2006 antisense SEQ ID NO: 2007 sense SEQ ID NO: 2008 antisense SEQ ID NO: 2009 sense SEQ ID NO: 2010 antisense SEQ ID NO: 2011 sense SEQ ID NO: 2012 antisense SEQ ID NO: 2013 sense SEQ ID NO: 2014 antisense SEQ ID NO: 2015 sense SEQ ID NO: 2016 antisense SEQ ID NO: 2017 sense SEQ ID NO: 2018 antisense SEQ ID NO: 2019 sense SEQ ID NO: 2020 antisense SEQ ID NO: 2021 sense SEQ ID NO: 2022 antisense SEQ ID NO: 2023 sense SEQ ID NO: 2024 antisense SEQ ID NO: 2025 sense SEQ ID NO: 2026 antisense SEQ ID NO: 2027 sense SEQ ID NO: 2028 antisense SEQ ID NO: 2029 sense SEQ ID NO: 2030 antisense SEQ ID NO: 2031 sense SEQ ID NO: 2032 antisense SEQ ID NO: 2033 sense SEQ ID NO: 2034 antisense SEQ ID NO: 2035 sense SEQ ID NO: 2036 antisense SEQ ID NO: 2037 sense SEQ ID NO: 2038 antisense SEQ ID NO: 2039 sense SEQ ID NO: 2040 antisense SEQ ID NO: 2041 sense SEQ ID NO: 2042 antisense SEQ ID NO: 2043 sense SEQ ID NO: 2044 antisense SEQ ID NO: 2045 sense SEQ ID NO: 2046 antisense SEQ ID NO: 2047 sense SEQ ID NO: 2048 antisense SEQ ID NO: 2049 sense SEQ ID NO: 2050 antisense SEQ ID NO: 2051 sense SEQ ID NO: 2052 antisense SEQ ID NO: 2053 sense SEQ ID NO: 2054 antisense SEQ ID NO: 2055 sense SEQ ID NO: 2056 antisense SEQ ID NO: 2057 sense SEQ ID NO: 2058 antisense SEQ ID NO: 2059 sense SEQ ID NO: 2060 antisense SEQ ID NO: 2061 sense SEQ ID NO: 2062 antisense SEQ ID NO: 2063 sense SEQ ID NO: 2064 antisense SEQ ID NO: 2065 sense SEQ ID NO: 2066 antisense SEQ ID NO: 2067 sense SEQ ID NO: 2068 antisense SEQ ID NO: 2069 sense SEQ ID NO: 2070 antisense SEQ ID NO: 2071 sense SEQ ID NO: 2072 antisense SEQ ID NO: 2073 sense SEQ ID NO: 2074 antisense SEQ ID NO: 2075 sense SEQ ID NO: 2076 antisense SEQ ID NO: 2077 sense SEQ ID NO: 2078 antisense SEQ ID NO: 2079 sense SEQ ID NO: 2080 antisense SEQ ID NO: 2081 sense SEQ ID NO: 2082 antisense SEQ ID NO: 2083 sense SEQ ID NO: 2084 antisense SEQ ID NO: 2085 sense SEQ ID NO: 2086 antisense SEQ ID NO: 2087 sense SEQ ID NO: 2088 antisense SEQ ID NO: 2089 sense SEQ ID NO: 2090 antisense SEQ ID NO: 2091 sense SEQ ID NO: 2092 antisense SEQ ID NO: 2093 sense SEQ ID NO: 2094 antisense SEQ ID NO: 2095 sense SEQ ID NO: 2096 antisense SEQ ID NO: 2097 sense SEQ ID NO: 2098 antisense SEQ ID NO: 2099 sense SEQ ID NO: 2100 antisense SEQ ID NO: 2101 sense SEQ ID NO: 2102 antisense SEQ ID NO: 2103 sense SEQ ID NO: 2104 antisense SEQ ID NO: 2105 sense SEQ ID NO: 2106 antisense SEQ ID NO: 2107 sense SEQ ID NO: 2108 antisense SEQ ID NO: 2109 sense SEQ ID NO: 2110 antisense SEQ ID NO: 2111 sense SEQ ID NO: 2112 antisense SEQ ID NO: 2113 sense SEQ ID NO: 2114 antisense SEQ ID NO: 2115 sense SEQ ID NO: 2116 antisense SEQ ID NO: 2117 sense SEQ ID NO: 2118 antisense SEQ ID NO: 2119 sense SEQ ID NO: 2120 antisense SEQ ID NO: 2121 sense SEQ ID NO: 2122 antisense SEQ ID NO: 2123 sense SEQ ID NO: 2124 antisense SEQ ID NO: 2125 sense SEQ ID NO: 2126 antisense SEQ ID NO: 2127 sense SEQ ID NO: 2128 antisense SEQ ID NO: 2129 sense SEQ ID NO: 2130 antisense SEQ ID NO: 2131 sense SEQ ID NO: 2132 antisense SEQ ID NO: 2133 sense SEQ ID NO: 2134 antisense SEQ ID NO: 2135 sense SEQ ID NO: 2136 antisense SEQ ID NO: 2137 sense SEQ ID NO: 2138 antisense SEQ ID NO: 2139 sense SEQ ID NO: 2140 antisense SEQ ID NO: 2141 sense SEQ ID NO: 2142 antisense SEQ ID NO: 2143 sense SEQ ID NO: 2144 antisense SEQ ID NO: 2145 sense SEQ ID NO: 2146 antisense SEQ ID NO: 2147 sense SEQ ID NO: 2148 antisense SEQ ID NO: 2149 sense SEQ ID NO: 2150 antisense SEQ ID NO: 2151 sense SEQ ID NO: 2152 antisense SEQ ID NO: 2153 sense SEQ ID NO: 2154 antisense SEQ ID NO: 2155 sense SEQ ID NO: 2156 antisense SEQ ID NO: 2157 sense SEQ ID NO: 2158 antisense SEQ ID NO: 2159 sense SEQ ID NO: 2160 antisense SEQ ID NO: 2161 sense SEQ ID NO: 2160 antisense SEQ ID NO: 2163 sense SEQ ID NO: 2164 antisense SEQ ID NO: 2165 sense SEQ ID NO: 2164 antisense SEQ ID NO: 2167 sense SEQ ID NO: 2168 antisense SEQ ID NO: 2169 sense SEQ ID NO: 2170 antisense SEQ ID NO: 2171 sense SEQ ID NO: 2172 antisense SEQ ID NO: 2173 sense SEQ ID NO: 2174 antisense SEQ ID NO: 2175 sense SEQ ID NO: 2176 antisense SEQ ID NO: 2177 sense SEQ ID NO: 2178 antisense SEQ ID NO: 2179 sense SEQ ID NO: 2180 antisense SEQ ID NO: 2181 sense SEQ ID NO: 2182 antisense SEQ ID NO: 2183 sense SEQ ID NO: 2184 antisense SEQ ID NO: 2185 sense SEQ ID NO: 2186 antisense SEQ ID NO: 2187 sense SEQ ID NO: 2188 antisense SEQ ID NO: 2189 sense SEQ ID NO: 2190 antisense SEQ ID NO: 2191 sense SEQ ID NO: 2192 antisense SEQ ID NO: 2193 sense SEQ ID NO: 2194 antisense SEQ ID NO: 2195 sense SEQ ID NO: 2196 antisense SEQ ID NO: 2197 sense SEQ ID NO: 2198 antisense SEQ ID NO: 2199 sense SEQ ID NO: 2200 antisense SEQ ID NO: 2201 sense SEQ ID NO: 2202 antisense SEQ ID NO: 2203 sense SEQ ID NO: 2204 antisense SEQ ID NO: 2205 sense SEQ ID NO: 2206 antisense SEQ ID NO: 2207 sense SEQ ID NO: 2208 antisense SEQ ID NO: 2209 sense SEQ ID NO: 2210 antisense SEQ ID NO: 2211 sense SEQ ID NO: 2212 antisense SEQ ID NO: 2213 sense SEQ ID NO: 2214 antisense SEQ ID NO: 2215 sense SEQ ID NO: 2216 antisense SEQ ID NO: 2217 sense SEQ ID NO: 2218 antisense SEQ ID NO: 2219 sense SEQ ID NO: 2220 antisense SEQ ID NO: 2221 sense SEQ ID NO: 2222 antisense SEQ ID NO: 2223 sense SEQ ID NO: 2224 antisense SEQ ID NO: 2225 sense SEQ ID NO: 2226 antisense SEQ ID NO: 2227 sense SEQ ID NO: 2228 antisense SEQ ID NO: 2229 sense SEQ ID NO: 2230 antisense SEQ ID NO: 2231 sense SEQ ID NO: 2232 antisense SEQ ID NO: 2233 sense SEQ ID NO: 2234 antisense SEQ ID NO: 2235 sense SEQ ID NO: 2236 antisense SEQ ID NO: 2237 sense SEQ ID NO: 2238 antisense SEQ ID NO: 2239 sense SEQ ID NO: 2240 antisense SEQ ID NO: 2241 sense SEQ ID NO: 2242 antisense SEQ ID NO: 2243 sense SEQ ID NO: 2244 antisense SEQ ID NO: 2245 sense SEQ ID NO: 2244 antisense SEQ ID NO: 2247 sense SEQ ID NO: 2248 antisense SEQ ID NO: 2249 sense SEQ ID NO: 2250 antisense SEQ ID NO: 2251 sense SEQ ID NO: 2252 antisense SEQ ID NO: 2253 sense SEQ ID NO: 2254 antisense SEQ ID NO: 2255 sense SEQ ID NO: 2256 antisense SEQ ID NO: 2257 sense SEQ ID NO: 2258 antisense SEQ ID NO: 2259 sense SEQ ID NO: 2260 antisense SEQ ID NO: 2261 sense SEQ ID NO: 2262 antisense SEQ ID NO: 2263 sense SEQ ID NO: 2264 antisense SEQ ID NO: 2265 sense SEQ ID NO: 2266 antisense SEQ ID NO: 2267 sense SEQ ID NO: 2268 antisense SEQ ID NO: 2269 sense SEQ ID NO: 2270 antisense SEQ ID NO: 2271 sense SEQ ID NO: 2272 antisense SEQ ID NO: 2273 sense SEQ ID NO: 2274 antisense SEQ ID NO: 2275 sense SEQ ID NO: 2276 antisense SEQ ID NO: 2277 sense SEQ ID NO: 2278 antisense SEQ ID NO: 2279 sense SEQ ID NO: 2280 antisense SEQ ID NO: 2281 sense SEQ ID NO: 2282 antisense SEQ ID NO: 2283 sense SEQ ID NO: 2284 antisense SEQ ID NO: 2285 sense SEQ ID NO: 2286 antisense SEQ ID NO: 2287 sense SEQ ID NO: 2288 antisense SEQ ID NO: 2289 sense SEQ ID NO: 2290 antisense SEQ ID NO: 2291 sense SEQ ID NO: 2292 antisense SEQ ID NO: 2293 sense SEQ ID NO: 2294 antisense SEQ ID NO: 2295 sense SEQ ID NO: 2296 antisense SEQ ID NO: 2353 sense SEQ ID NO: 2354 antisense SEQ ID NO: 2355 sense SEQ ID NO: 2356 antisense SEQ ID NO: 2357 sense SEQ ID NO: 2358 antisense SEQ ID NO: 2359 sense SEQ ID NO: 2360 antisense SEQ ID NO: 2361 sense SEQ ID NO: 2362 antisense SEQ ID NO: 2363 sense SEQ ID NO: 2364 antisense SEQ ID NO: 2365 sense SEQ ID NO: 2364 antisense SEQ ID NO: 2367 sense SEQ ID NO: 2368 antisense SEQ ID NO: 2369 sense SEQ ID NO: 2370 antisense SEQ ID NO: 2371 sense SEQ ID NO: 2372 antisense SEQ ID NO: 2373 sense SEQ ID NO: 2374 antisense SEQ ID NO: 2375 sense SEQ ID NO: 2376 antisense SEQ ID NO: 2377 sense SEQ ID NO: 2378 antisense SEQ ID NO: 2379 sense SEQ ID NO: 2380 antisense SEQ ID NO: 2381 sense SEQ ID NO: 2382 antisense SEQ ID NO: 2383 sense SEQ ID NO: 2384 antisense SEQ ID NO: 2385 sense SEQ ID NO: 2386 antisense SEQ ID NO: 2387 sense SEQ ID NO: 2388 antisense SEQ ID NO: 2389 sense SEQ ID NO: 2390 antisense SEQ ID NO: 2391 sense SEQ ID NO: 2392 antisense SEQ ID NO: 2393 sense SEQ ID NO: 2394 antisense SEQ ID NO: 2395 sense SEQ ID NO: 2396 antisense SEQ ID NO: 2397 sense SEQ ID NO: 2398 antisense SEQ ID NO: 2399 sense SEQ ID NO: 2400 antisense SEQ ID NO: 2401 sense SEQ ID NO: 2402 antisense SEQ ID NO: 2403 sense SEQ ID NO: 2404 antisense SEQ ID NO: 2405 sense SEQ ID NO: 2406 antisense SEQ ID NO: 2407 sense SEQ ID NO: 2408 antisense SEQ ID NO: 2409 sense SEQ ID NO: 2410 antisense SEQ ID NO: 2411 sense SEQ ID NO: 2412 antisense SEQ ID NO: 2413 sense SEQ ID NO: 2414 antisense SEQ ID NO: 2415 sense SEQ ID NO: 2416 antisense SEQ ID NO: 2417 sense SEQ ID NO: 2418 antisense SEQ ID NO: 2419 sense SEQ ID NO: 2420 antisense SEQ ID NO: 2421 sense SEQ ID NO: 2422 antisense SEQ ID NO: 2423 sense SEQ ID NO: 2424 antisense SEQ ID NO: 2425 sense SEQ ID NO: 2426 antisense SEQ ID NO: 2427 sense SEQ ID NO: 2428 antisense SEQ ID NO: 2429 sense SEQ ID NO: 2430 antisense SEQ ID NO: 2431 sense SEQ ID NO: 2432 antisense SEQ ID NO: 2433 sense SEQ ID NO: 2434 antisense SEQ ID NO: 2435 sense SEQ ID NO: 2436 antisense SEQ ID NO: 2437 sense SEQ ID NO: 2438 antisense SEQ ID NO: 2439 sense SEQ ID NO: 2440 antisense SEQ ID NO: 2441 sense SEQ ID NO: 2442 antisense SEQ ID NO: 2443 sense SEQ ID NO: 2444 antisense SEQ ID NO: 2445 sense SEQ ID NO: 2446 antisense SEQ ID NO: 2447 sense SEQ ID NO: 2448 antisense SEQ ID NO: 2449 sense SEQ ID NO: 2450 antisense SEQ ID NO: 2451 sense SEQ ID NO: 2452 antisense SEQ ID NO: 2453 sense SEQ ID NO: 2454 antisense SEQ ID NO: 2455 sense SEQ ID NO: 2456 antisense SEQ ID NO: 2457 sense SEQ ID NO: 2458 antisense SEQ ID NO: 2459 sense SEQ ID NO: 2460 antisense SEQ ID NO: 2461 sense SEQ ID NO: 2462 antisense SEQ ID NO: 2463 sense SEQ ID NO: 2464 antisense SEQ ID NO: 2465 sense SEQ ID NO: 2466 antisense SEQ ID NO: 2467 sense SEQ ID NO: 2468 antisense SEQ ID NO: 2469 sense SEQ ID NO: 2470 antisense SEQ ID NO: 2471 sense SEQ ID NO: 2472 antisense SEQ ID NO: 2473 sense SEQ ID NO: 2474 antisense SEQ ID NO: 2475 sense SEQ ID NO: 2476 antisense SEQ ID NO: 2477 sense SEQ ID NO: 2478 antisense SEQ ID NO: 2479 sense SEQ ID NO: 2480 antisense SEQ ID NO: 2481 sense SEQ ID NO: 2482 antisense SEQ ID NO: 2483 sense SEQ ID NO: 2484 antisense SEQ ID NO: 2485 sense SEQ ID NO: 2486 antisense SEQ ID NO: 2487 sense SEQ ID NO: 2488 antisense SEQ ID NO: 2489 sense SEQ ID NO: 2490 antisense SEQ ID NO: 2491 sense SEQ ID NO: 2492 antisense SEQ ID NO: 2493 sense SEQ ID NO: 2494 antisense SEQ ID NO: 2495 sense SEQ ID NO: 2496 antisense SEQ ID NO: 2497 sense SEQ ID NO: 2498 antisense SEQ ID NO: 2499 sense SEQ ID NO: 2500 antisense SEQ ID NO: 2501 sense SEQ ID NO: 2502 antisense SEQ ID NO: 2503 sense SEQ ID NO: 2504 antisense SEQ ID NO: 2505 sense SEQ ID NO: 2506 antisense SEQ ID NO: 2507 sense SEQ ID NO: 2508 antisense SEQ ID NO: 2509 sense SEQ ID NO: 2510 antisense SEQ ID NO: 2511 sense SEQ ID NO: 2512 antisense SEQ ID NO: 2513 sense SEQ ID NO: 2514 antisense SEQ ID NO: 2515 sense SEQ ID NO: 2516 antisense SEQ ID NO: 2573 sense SEQ ID NO: 2574 antisense SEQ ID NO: 2575 sense SEQ ID NO: 2576 antisense SEQ ID NO: 2577 sense SEQ ID NO: 2578 antisense SEQ ID NO: 2579 sense SEQ ID NO: 2580 antisense SEQ ID NO: 2581 sense SEQ ID NO: 2582 antisense SEQ ID NO: 2583 sense SEQ ID NO: 2584 antisense SEQ ID NO: 2585 sense SEQ ID NO: 2586 antisense SEQ ID NO: 2587 sense SEQ ID NO: 2588 antisense SEQ ID NO: 2589 sense SEQ ID NO: 2590 antisense SEQ ID NO: 2591 sense SEQ ID NO: 2592 antisense SEQ ID NO: 2593 sense SEQ ID NO: 2594 antisense SEQ ID NO: 2595 sense SEQ ID NO: 2596 antisense SEQ ID NO: 2597 sense SEQ ID NO: 2598 antisense SEQ ID NO: 2599 sense SEQ ID NO: 2600 antisense SEQ ID NO: 2601 sense SEQ ID NO: 2602 antisense SEQ ID NO: 2603 sense SEQ ID NO: 2604 antisense SEQ ID NO: 2605 sense SEQ ID NO: 2606 antisense SEQ ID NO: 2607 sense SEQ ID NO: 2608 antisense SEQ ID NO: 2609 sense SEQ ID NO: 2610 antisense SEQ ID NO: 2611 sense SEQ ID NO: 2612 antisense SEQ ID NO: 2613 sense SEQ ID NO: 2614 antisense SEQ ID NO: 2615 sense SEQ ID NO: 2616 antisense SEQ ID NO: 2617 sense SEQ ID NO: 2618 antisense SEQ ID NO: 2619 sense SEQ ID NO: 2620 antisense SEQ ID NO: 2621 sense SEQ ID NO: 2622 antisense SEQ ID NO: 2623 sense SEQ ID NO: 2624 antisense SEQ ID NO: 2625 sense SEQ ID NO: 2626 antisense SEQ ID NO: 2627 sense SEQ ID NO: 2628 antisense SEQ ID NO: 2629 sense SEQ ID NO: 2630 antisense SEQ ID NO: 2631 sense SEQ ID NO: 2632 antisense SEQ ID NO: 2633 sense SEQ ID NO: 2634 antisense SEQ ID NO: 2635 sense SEQ ID NO: 2636 antisense SEQ ID NO: 2637 sense SEQ ID NO: 2638 antisense SEQ ID NO: 2639 sense SEQ ID NO: 2640 antisense SEQ ID NO: 2641 sense SEQ ID NO: 2642 antisense SEQ ID NO: 2643 sense SEQ ID NO: 2644 antisense SEQ ID NO: 2645 sense SEQ ID NO: 2646 antisense SEQ ID NO: 2647 sense SEQ ID NO: 2648 antisense SEQ ID NO: 2649 sense SEQ ID NO: 2650 antisense SEQ ID NO: 2651 sense SEQ ID NO: 2652 antisense SEQ ID NO: 2653 sense SEQ ID NO: 2654 antisense SEQ ID NO: 2655 sense SEQ ID NO: 2656 antisense SEQ ID NO: 2657 sense SEQ ID NO: 2650 antisense SEQ ID NO: 2659 sense SEQ ID NO: 2660 antisense SEQ ID NO: 2661 sense SEQ ID NO: 2662 antisense SEQ ID NO: 2663 sense SEQ ID NO: 2664 antisense SEQ ID NO: 2665 sense SEQ ID NO: 2666 antisense SEQ ID NO: 2667 sense SEQ ID NO: 2668 antisense SEQ ID NO: 2669 sense SEQ ID NO: 2670 antisense SEQ ID NO: 2671 sense SEQ ID NO: 2672 antisense SEQ ID NO: 2673 sense SEQ ID NO: 2674 antisense SEQ ID NO: 2675 sense SEQ ID NO: 2676 antisense SEQ ID NO: 2677 sense SEQ ID NO: 2678 antisense SEQ ID NO: 2679 sense SEQ ID NO: 2680 antisense SEQ ID NO: 2681 sense SEQ ID NO: 2682 antisense SEQ ID NO: 2683 sense SEQ ID NO: 2684 antisense SEQ ID NO: 2685 sense SEQ ID NO: 2686 antisense SEQ ID NO: 2687 sense SEQ ID NO: 2688 antisense SEQ ID NO: 2689 sense SEQ ID NO: 2690 antisense SEQ ID NO: 2691 sense SEQ ID NO: 2692 antisense SEQ ID NO: 2693 sense SEQ ID NO: 2694 antisense SEQ ID NO: 2695 sense SEQ ID NO: 2696 antisense SEQ ID NO: 2697 sense SEQ ID NO: 2698 antisense SEQ ID NO: 2699 sense SEQ ID NO: 2700 antisense SEQ ID NO: 2701 sense SEQ ID NO: 2702 antisense SEQ ID NO: 2703 sense SEQ ID NO: 2704 antisense SEQ ID NO: 2705 sense SEQ ID NO: 2706 antisense SEQ ID NO: 2707 sense SEQ ID NO: 2708 antisense SEQ ID NO: 2709 sense SEQ ID NO: 2710 antisense SEQ ID NO: 2711 sense SEQ ID NO: 2712 antisense SEQ ID NO: 2713 sense SEQ ID NO: 2714 antisense SEQ ID NO: 2715 sense SEQ ID NO: 2716 antisense SEQ ID NO: 2717 sense SEQ ID NO: 2718 antisense SEQ ID NO: 2719 sense SEQ ID NO: 2720 antisense SEQ ID NO: 2721 sense SEQ ID NO: 2722 antisense SEQ ID NO: 2723 sense SEQ ID NO: 2724 antisense SEQ ID NO: 2725 sense SEQ ID NO: 2726 antisense SEQ ID NO: 2727 sense SEQ ID NO: 2728 antisense SEQ ID NO: 2729 sense SEQ ID NO: 2730 antisense SEQ ID NO: 2731 sense SEQ ID NO: 2732 antisense SEQ ID NO: 2733 sense SEQ ID NO: 2734 antisense SEQ ID NO: 2735 sense SEQ ID NO: 2736 antisense SEQ ID NO: 2737 sense SEQ ID NO: 2738 antisense SEQ ID NO: 2739 sense SEQ ID NO: 2740 antisense SEQ ID NO: 2741 sense SEQ ID NO: 2742 antisense SEQ ID NO: 2743 sense SEQ ID NO: 2744 antisense SEQ ID NO: 2745 sense SEQ ID NO: 2746 antisense SEQ ID NO: 2747 sense SEQ ID NO: 2748 antisense SEQ ID NO: 2749 sense SEQ ID NO: 2750 antisense SEQ ID NO: 2751 sense SEQ ID NO: 2752 antisense SEQ ID NO: 2753 sense SEQ ID NO: 2754 antisense SEQ ID NO: 2755 sense SEQ ID NO: 2756 antisense SEQ ID NO: 2757 sense SEQ ID NO: 2758 antisense SEQ ID NO: 2759 sense SEQ ID NO: 2760 antisense SEQ ID NO: 2761 sense SEQ ID NO: 2762 antisense SEQ ID NO: 2763 sense SEQ ID NO: 2764 antisense SEQ ID NO: 2765 sense SEQ ID NO: 2766 antisense SEQ ID NO: 2767 sense SEQ ID NO: 2768 antisense SEQ ID NO: 2769 sense SEQ ID NO: 2770 antisense SEQ ID NO: 2771 sense SEQ ID NO: 2772 antisense SEQ ID NO: 2773 sense SEQ ID NO: 2774 antisense SEQ ID NO: 2775 sense SEQ ID NO: 2776 antisense SEQ ID NO: 2777 sense SEQ ID NO: 2778 antisense SEQ ID NO: 2779 sense SEQ ID NO: 2780 antisense SEQ ID NO: 2781 sense SEQ ID NO: 2782 antisense SEQ ID NO: 2783 sense SEQ ID NO: 2784 antisense SEQ ID NO: 2785 sense SEQ ID NO: 2786 antisense SEQ ID NO: 2787 sense SEQ ID NO: 2788 antisense SEQ ID NO: 2789 sense SEQ ID NO: 2790 antisense SEQ ID NO: 2791 sense SEQ ID NO: 2792 antisense

TABLE 7 Transforming growth factor beta iRNA sequences Sense and Corresponding Antisense Duplexes Sense and Corresponding Antisense Duplexes Corresponding Corresponding Sense Strand Antisense Strand Sense Strand Antisense Strand SEQ ID NO: 2793 sense SEQ ID NO: 2794 antisense SEQ ID NO: 2795 sense SEQ ID NO: 2796 sense SEQ ID NO: 2797 sense SEQ ID NO: 2798 antisense SEQ ID NO: 2799 sense SEQ ID NO: 2800 sense SEQ ID NO: 2801 sense SEQ ID NO: 2802 antisense SEQ ID NO: 2803 sense SEQ ID NO: 2804 sense SEQ ID NO: 2805 sense SEQ ID NO: 2806 antisense SEQ ID NO: 2807 sense SEQ ID NO: 2808 sense SEQ ID NO: 2809 sense SEQ ID NO: 2810 antisense SEQ ID NO: 2811 sense SEQ ID NO: 2812 sense SEQ ID NO: 2813 sense SEQ ID NO: 2814 antisense SEQ ID NO: 2815 sense SEQ ID NO: 2816 sense SEQ ID NO: 2817 sense SEQ ID NO: 2818 antisense SEQ ID NO: 2819 sense SEQ ID NO: 2820 sense SEQ ID NO: 2821 sense SEQ ID NO: 2822 antisense SEQ ID NO: 2823 sense SEQ ID NO: 2824 sense SEQ ID NO: 2825 sense SEQ ID NO: 2826 antisense SEQ ID NO: 2827 sense SEQ ID NO: 2828 sense SEQ ID NO: 2829 sense SEQ ID NO: 2830 antisense SEQ ID NO: 2831 sense SEQ ID NO: 2832 sense SEQ ID NO: 2833 sense SEQ ID NO: 2834 antisense SEQ ID NO: 2835 sense SEQ ID NO: 2836 sense SEQ ID NO: 2837 sense SEQ ID NO: 2838 antisense SEQ ID NO: 2839 sense SEQ ID NO: 2840 sense SEQ ID NO: 2841 sense SEQ ID NO: 2842 antisense SEQ ID NO: 2843 sense SEQ ID NO: 2844 sense SEQ ID NO: 2845 sense SEQ ID NO: 2846 antisense SEQ ID NO: 2847 sense SEQ ID NO: 2848 sense SEQ ID NO: 2849 sense SEQ ID NO: 2850 antisense SEQ ID NO: 2851 sense SEQ ID NO: 2852 sense SEQ ID NO: 2853 sense SEQ ID NO: 2854 antisense SEQ ID NO: 2855 sense SEQ ID NO: 2856 sense SEQ ID NO: 2857 sense SEQ ID NO: 2858 antisense SEQ ID NO: 2859 sense SEQ ID NO: 2860 sense

TABLE 8 Commonly used liver fibrosis staging scores. Staging System Fibrosis stages Remarks METAVIR Score F0, F1, F2, Best evaluated in (The French METAVIR F3, F4 HCV fibrosis Cooperative Study Group 1994) Knodell Score F0, F1, F3, F4 No intermediate stage (Knodell 1981) Desmet & Scheuer Analogous Recommended by (Desmet 1994: to METAVIR German guidelines Schirmacher 2004) for the assessment of liver fibrosis Batts & Ludwigg Similar to (Batts 1995) METAVIR Ishak Score F0, F1, F2, F3, (Ishak 1995) F4, F5, F6

-   Batts K P, Ludwig J. Chronic hepatitis. An update on terminology and     reporting. Am J Surg Pathol 1995, 19(12):1409-17. -   Desmet V J, Gerber M, Hoofnagle J H, Manns M, Scheuer P J.     Classification of chronic hepatitis: diagnosis, grading and staging.     Hepatology 1994, 19(6):1513-20. -   Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, et     al. Histological grading and staging of chronic hepatitis. J Hepatol     1995, 22(6):696-9. -   Knodell R G, Ishak K G, Black W C, Chen T S, Craig R, Kaplowitz N,     Kiernan T W, Wollman J. Formulation and application of a numerical     scoring system for assessing histological activity in asymptomatic     chronic active hepatitis. Hepatology 1981, 1(5):431-5. -   Schirmacher P, Fleig W E, Dienes H P. (Biopsy diagnosis of chronic     hepatitis). Z Gastroenterol 2004, 42(2):175-85.

TABLE 9 Common biomarkers of liver fibrosis. Index Markers Calculation Interpretation PPV/NPV (%) Direct surrogate markers MP3 PII|NP, MMP-1 0.5901(logPI|NP[ng/ml]) − <0.3 = F0-2 NPV = 95 0.1749(logMMP-1[ng/ml]) >0.4 = F3-4 PPV = 66 <0.3 = F0-1 NPV = 75 >0.4 = F2-4 PPV = 91 ELF PII|NP, HA Proprietary >0.102 Scheuer 3-4 PPV = 35 <0.102 Scheuer 0-2 NPV = 92 Indirect surrogate markers Forms Age, pit, γGT, 7.811 − 3.131 × in(pit) + >6.9 = Scheuer 2-4 PPV = 66 cholesterol 0.781 × in(γGT) + 3.467 <4.2 = Scheuer 0-1 NPV = 96 In(age) − 0.014 (cholesterol) APRI AST, pit ([AST/ULN]/pit >1.5 = |shak 3-6 PPV = 91 [× 109/l]) × 100 ≤0.5 = |shak 0-2 NPV = 90 Fibrotest Haptoglobin, Proprietary 0.75-1.00 = F4 PPV = 78 Fibrosure a2-MC, 0.73-0.74 = F3-F4 PPV = 76 apo-A1, γGT, 0.59-0.72 = F3 PPV = 76 bilirubin, 0.49-0.58 = F2 PPV = 67 γ-globulin 0.32-0.48 = F1-F2 PPV/NPV = 61/85 0.28-0.31 = F1 NPV = 91 0.22-0.27 = F0-F1 NPV = 92 0.00-0.21 = F0 NPV = 94 Fibroindex Pit, AST, γGT 1.736 − 0.064 (pit [¥104/mm3]) + ≤1.25 = F0-F1 NPV = 61.7 0.005 (AST [IU/L]) + 0.463 × ≥2.25 = F2-F3 PPV = 90 (γGT(g/dl) Testa Pit, spleen Pit count/spleen diameter >1750 = |shak ≤2 NPV = 79 diameter ≤1750 = |shak <2 PPV = 78.9c Fibrosis AST, cholesterol, E*/1 + e*, where * = −10.929 + <0.2 = F0-F1 NPV = 77.4 probability past alcohol (1.827 × in[AST]) + (0.081 × age) + ≥0.8 = F2-F4 PPV = 87 index intake, HOMA, age (0.768 × [past alcohol use graded as 0-2]) + (0.385 × HOMA) FIB-4 Pit, AST, ALT, age (Age × AST)/(pit count × ALT1/2) <1.45 = |shak <4-6 NPV = 90 >3.25 = |shak ≥4-6 PPV = 65 Bonancini ALT, AST ,INR, pit Sum (range 0-11) of (pit score) + >8 = Knodell 3-4 PPV = 92.9 (ALT/AST score) + (INR score) pit (× 109/l): >340 = 0; 280-339 = 1; 220-279 = 2; 160-219 = 3; 100-159 = 4; 40-99 = 5; <40 = 6 ALT/AST ratio; >1.7 = 0; 1.27-1.7 = 1; 0.6-1.19 = 2; <0.6 = 3 INR; \1.4 = 2 Pohl AST, ALT, pit Positive if: AST/ALT ≥1 and platelet Positive = F3-F4 PPV = 93 count <150 × 109/l Shet AST, ALT AST/ALT ≥1 = Scheuer 4 PPV = 100 Park AST, ALT AST/ALT ≥1 = Scheuer 4 PPV = 73.7 Age-Platelet Pit, age Age score + pit score (0-10 possible ≥6 = F2-F4 PPV = 96 score) age: <30 = 0; 30-39 = 1; 40-49 = 2; 50-59 = 3; 60-69 = 4; ≥70 = 5. Pit (× 109/l): ≥225 = 0; 200-224 = 1; 175-199 = 2; 150-174 = 3; 125-149 = 4; <125 = 5 Combined direct and indirect surrogate markers SHASTA HA, AST, albumin −384 + 1.70 (1 if HA 41-85 ng/ml, 0 >0.8 = |shak ≥3 PPV = 100 otherwise) + 3.28 (1 if HA >85 ng/ml, <0.3 = |shak ≤2 NPV = 94 0 otherwise) + 1.58 (1 if albumin <3.5 g/dl, 0 otherwise) + 1.78 (1 if AST >60 IU/l, 0 otherwise) FM pit, PI, AST, HA, −0.007 pit (G/L) − 0.049 PI (%) + 0.012 ≥F2 PPV = 86.3/96.6 α2-MC, gender, AST (IU/l) + 0.005 α2-MC (mg/dl) + 0.021 age HA ( g/l) − 0.270 urea (mmol/l) + 0.027 age (years) + 3.718 Hepascore HA, α2-MC, γGT, y/1 + y, where y = exp[−4.185818 − ≥0.5 = F2-F4 PPV = 88 age, gender (0.0249 × age) + 0.7464 × sex) + (1.0039 × <0.5 = F0-F1 NPV = 98 α2-MC) + (0.0302 × HA) + (0.0691 × bilirubin) − (0.0012 × γGT)] FSII Ha, α2-MC, TIMP-1 Proprietary ≥42 = F2-F4 PPV = 77.4 <40 = F0-F1 NPV = 78 

1.-4. (canceled)
 5. A double-stranded ribonucleic acid (dsRNA) for inhibiting expression of TGFβ, wherein said dsRNA comprises a sense strand and an antisense strand, the sense strand comprising one of SEQ ID NOs: 67, 71, 73, 87, 89, 107, 109, 111, 125 and 127, and the antisense strand comprising, respectively, one of SEQ ID NOs: 68, 72, 74, 88, 90, 108, 110, 112, 126 and
 128. 6.-7. (canceled)
 8. The dsRNA of claim 5, wherein said dsRNA comprises at least one modified nucleotide.
 9. The dsRNA of claim 8, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. 10.-11. (canceled)
 12. The dsRNA of claim 5, wherein each strand is no more than 30 nucleotides in length.
 13. The dsRNA of claim 5, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 14. The dsRNA of claim 5, further comprising a ligand.
 15. The dsRNA of claim 14, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA. 16.-21. (canceled)
 22. A cell containing the dsRNA of any of claim
 5. 23. (canceled)
 24. A pharmaceutical composition for inhibiting expression of a TGFβ gene comprising the dsRNA of claim
 5. 25. (canceled)
 26. A lipid formulation comprising a dsRNA of claim
 5. 27. The lipid formulation of claim 26, wherein the lipid formulation is a SNALP, or XTC formulation.
 28. A method of inhibiting TGFβ expression in a cell, the method comprising: a. introducing into the cell the dsRNA of claim 5; and b. maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TGFβ gene, thereby inhibiting expression of the TGFβ gene in the cell.
 29. The method of claim 28, wherein the cell is a hepatic stellate cell.
 30. The method of claim 28, wherein the TGFβ expression is inhibited by at least 30%.
 31. A method of treating liver fibrosis comprising administering to a human in need of such treatment a therapeutically effective amount of the dsRNA of claim
 5. 32. The method of claim 31, wherein the dsRNA is administered at a concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject. 33.-34. (canceled)
 35. A vector encoding a dsRNA, wherein said dsRNA comprises a sense strand and an antisense strand, the sense strand comprising one of SEQ ID NOs: 67, 71, 73, 87, 89, 107, 109, 111, 125 and 127, and the antisense strand comprising, respectively, one of SEQ ID NOs: 68, 72, 74, 88, 90, 108, 110, 112, 126 and
 128. 36.-37. (canceled)
 38. A cell comprising the vector of claim
 35. 39.-40. (canceled)
 41. The double-stranded ribonucleic acid of claim 5, wherein the sense strand comprises nucleotide sequence of SEQ ID NO: 71 and the antisense strand comprises nucleotide sequence of SEQ ID NO:
 72. 42. The double-stranded ribonucleic acid of claim 5, wherein the sense strand comprises nucleotide sequence of SEQ ID NO: 107 and the antisense strand comprises nucleotide sequence of SEQ ID NO:
 108. 